Regulation of Growth and Metastases in an Estrogen Independent Breast Cancer Cell by Vitamin D Compounds

DTIC Science & Technology

2000-08-01

micrometastases in the nude mouse, we used the EGFP fluorescent marker gene, cloned from the bioluminescent jellyfish Aequorea Victoria (previously...Preparation. "* Flanagan L, Byrne I, Wang Y, Tenniswood M and Welsh JE. Utilization of green fluorescent protein for the identification of metastasis...phenotype. Utilizing the SUM-159PT cell line stably transfected with pEGFP-C1 (enhanced green fluorescent protein ) we have been able to successfully track

Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent.

PubMed

Chernousova, S; Epple, M

2017-05-01

The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2-3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application.

Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent

PubMed Central

Chernousova, S; Epple, M

2017-01-01

The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2–3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application. PMID:28218744

[Establishment of a human bladder cancer cell line stably co-expressing hSPRY2 and luciferase genes and its subcutaneous tumor xenograft model in nude mice].

PubMed

Yin, Xiaotao; Li, Fanglong; Jin, Yipeng; Yin, Zhaoyang; Qi, Siyong; Wu, Shuai; Wang, Zicheng; Wang, Lin; Yu, Jiyun; Gao, Jiangping

2017-03-01

Objective To establish a human bladder cancer cell line stably co-expressing human sprouty2 (hSPRY2) and luciferase (Luc) genes simultaneously, and develop its subcutaneous tumor xenograft model in nude mice. Methods The hSPRY2 and Luc gene segments were amplified by PCR, and were cloned into lentiviral vector pCDH and pLVX respectively to produce corresponding lentivirus particles. The J82 human bladder cancer cells were infected with these two kinds of lentivirus particles, and then further screened by puromycin and G418. The expressions of hSPRY2 and Luc genes were detected by bioluminescence, immunofluorescence and Western blot analysis. The screened J82-hSPRY2/Luc cells were injected subcutaneously into BALB/c nude mice, and the growth of tumor was monitored dynamically using in vivo fluorescence imaging system. Results J82-hSPRY2/Luc cell line stably expressing hSPRY2 and Luc genes was established successfully. Bioluminescence, immunofluorescence and Western blot analysis validated the expressions of hSPRY2 and Luc genes. The in vivo fluorescence imaging system showed obvious fluorescence in subcutaneous tumor xenograft in nude mice. Conclusion The J82-hSPRY2/Luc bladder cancer cell line and its subcutaneous tumor xenograft model in nude mice have been established successfully.

Bioluminescence in the Sea

NASA Astrophysics Data System (ADS)

Haddock, Steven H. D.; Moline, Mark A.; Case, James F.

2010-01-01

Bioluminescence spans all oceanic dimensions and has evolved many times—from bacteria to fish—to powerfully influence behavioral and ecosystem dynamics. New methods and technology have brought great advances in understanding of the molecular basis of bioluminescence, its physiological control, and its significance in marine communities. Novel tools derived from understanding the chemistry of natural light-producing molecules have led to countless valuable applications, culminating recently in a related Nobel Prize. Marine organisms utilize bioluminescence for vital functions ranging from defense to reproduction. To understand these interactions and the distributions of luminous organisms, new instruments and platforms allow observations on individual to oceanographic scales. This review explores recent advances, including the chemical and molecular, phylogenetic and functional, community and oceanographic aspects of bioluminescence.

Developing a Predictive Capability for Bioluminescence Signatures

DTIC Science & Technology

2012-09-30

sources of bioluminescence are dinoflagellates, common unicellular plankton that are also known to form red tides. Dinoflagellate bioluminescence is...to locate their prey. The bioluminescence signature of a moving object depends on the bioluminescence potential of the organisms (related to...characteristics of the source organisms and the measurement of their bioluminescence by ocean Report Documentation Page Form ApprovedOMB No. 0704-0188 Public

Assessing laser-tissue damage with bioluminescent imaging

NASA Astrophysics Data System (ADS)

Wilmink, Gerald J.; Opalenik, Susan R.; Beckham, Josh T.; Davidson, Jeffrey M.; Jansen, Eric D.

2006-07-01

Effective medical laser procedures are achieved by selecting laser parameters that minimize undesirable tissue damage. Traditionally, human subjects, animal models, and monolayer cell cultures have been used to study wound healing, tissue damage, and cellular effects of laser radiation. Each of these models has significant limitations, and consequently, a novel skin model is needed. To this end, a highly reproducible human skin model that enables noninvasive and longitudinal studies of gene expression was sought. In this study, we present an organotypic raft model (engineered skin) used in combination with bioluminescent imaging (BLI) techniques. The efficacy of the raft model was validated and characterized by investigating the role of heat shock protein 70 (hsp70) as a sensitive marker of thermal damage. The raft model consists of human cells incorporated into an extracellular matrix. The raft cultures were transfected with an adenovirus containing a murine hsp70 promoter driving transcription of luciferase. The model enables quantitative analysis of spatiotemporal expression of proteins using BLI. Thermal stress was induced on the raft cultures by means of a constant temperature water bath or with a carbon dioxide (CO2) laser (λ=10.6 µm, 0.679 to 2.262 W/cm2, cw, unfocused Gaussian beam, ωL=4.5 mm, 1 min exposure). The bioluminescence was monitored noninvasively with an IVIS 100 Bioluminescent Imaging System. BLI indicated that peak hsp70 expression occurs 4 to 12 h after exposure to thermal stress. A minimum irradiance of 0.679 W/cm2 activated the hsp70 response, and a higher irradiance of 2.262 W/cm2 was associated with a severe reduction in hsp70 response due to tissue ablation. Reverse transcription polymerase chain reaction demonstrated that hsp70 mRNA levels increased with prolonged heating exposures. Enzyme-linked immunosorbent protein assays confirmed that luciferase was an accurate surrogate for hsp70 intracellular protein levels. Hematoxylin and

Bathyphotometer bioluminescence potential measurements: A framework for characterizing flow agitators and predicting flow-stimulated bioluminescence intensity

NASA Astrophysics Data System (ADS)

Latz, Michael I.; Rohr, Jim

2013-07-01

Bathyphotometer measurements of bioluminescence are used as a proxy for the abundance of luminescent organisms for studying population dynamics; the interaction of luminescent organisms with physical, chemical, and biological oceanographic processes; and spatial complexity especially in coastal areas. However, the usefulness of bioluminescence measurements has been limited by the inability to compare results from different bathyphotometer designs, or even the same bathyphotometer operating at different volume flow rates. The primary objective of this study was to compare measurements of stimulated bioluminescence of four species of cultured dinoflagellates, the most common source of bioluminescence in coastal waters, using two different bathyphotometer flow agitators as a function of bathyphotometer volume flow rate and dinoflagellate concentration. For both the NOSC and BIOLITE flow agitators and each species of dinoflagellate tested, there was a critical volume flow rate, above which average bioluminescence intensity, designated as bathyphotometer bioluminescence potential (BBP), remained relatively constant and scaled directly with dinoflagellate cell concentration. At supra-critical volume flow rates, the ratio of BIOLITE to NOSC BBP was nearly constant for the same species studied, but varied between species. The spatial pattern and residence time of flash trajectories within the NOSC flow agitator indicated the presence of dominant secondary recirculating flows, where most of the bioluminescence was detected. A secondary objective (appearing in the Appendix) was to study the feasibility of using NOSC BBP to scale flow-stimulated bioluminescence intensity across similar flow fields, where the contributing composition of luminescent species remained the same. Fully developed turbulent pipe flow was chosen because it is hydrodynamically well characterized. Average bioluminescence intensity in a 2.54-cm i.d. pipe was highly correlated with wall shear stress and

Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells.

PubMed

Higashimoto, Yuji; Elliott, W Mark; Behzad, Ali R; Sedgwick, Edward G; Takei, Tatsuo; Hogg, James C; Hayashi, Shizu

2002-07-15

Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells.

PubMed

Fang, Mingzhu; Kang, Hwan-Goo; Park, Youngil; Estrella, Brian; Zarbl, Helmut

2017-09-28

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both

A transfected cell model for the renal toxin transporter, rOCT2.

PubMed

Pan, B F; Sweet, D H; Pritchard, J B; Chen, R; Nelson, J A

1999-02-01

A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied.

Plasma-mediated transfection of RPE

NASA Astrophysics Data System (ADS)

Palanker, D.; Chalberg, T.; Vankov, A.; Huie, P.; Molnar, F. E.; Butterwick, A.; Calos, M.; Marmor, M.; Blumenkranz, M. S.

2006-02-01

A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery techniques. Viral delivery has encountered a number of serious problems including immunological reactions and malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo or produce severe collateral damage to ocular tissues. We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation. For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient transfection of RPE without visible damage to the retina. Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor 10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.

Developing a Predictive Capability for Bioluminescence Signatures

DTIC Science & Technology

2010-01-01

sources of bioluminescence are dinoflagellates, common unicellular plankton that are also known to form red tides. Dinoflagellate bioluminescence is...wakes to locate their prey. The bioluminescence signature of a moving object depends on the bioluminescence potential of the organisms (related...PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) University of California, San Diego,Scripps Institution of Oceanography,La Jolla,CA,92093-0202 8

Imaging of bioluminescent LNCaP-luc-M6 tumors: a new animal model for the study of metastatic human prostate cancer.

PubMed

Scatena, Caroline D; Hepner, Mischa A; Oei, Yoko A; Dusich, Joan M; Yu, Shang-Fan; Purchio, Tony; Contag, Pamela R; Jenkins, Darlene E

2004-05-15

Animal experiments examining hormone-sensitive metastatic prostate cancer using the human LNCaP cell line have been limited to endpoint analyses. To permit longitudinal studies, we generated a luciferase-expressing cell line and used bioluminescent imaging (BLI) to non-invasively monitor the in vivo growth of primary LNCaP tumors and metastasis. LNCaP.FGC cells were transfected to constitutively express firefly luciferase. LNCaP-luc-M6 cells were tested for bioluminescent signal intensity and hormone responsiveness in vitro. The cells were implanted in subcutaneous and orthotopic sites in SCID-bg mice and imaged over time. The LNCaP-luc-M6 cells formed subcutaneous and orthotopic tumors in SCID-bg mice, and nearly all tumor-bearing animals developed pulmonary metastases. Early detection and temporal growth of primary tumors and metastatic lesions was successfully monitored by BLI. The LNCaP-luc-M6 cell line is a bioluminescent, hormone-sensitive prostate cancer cell line applicable for BLI studies to non-invasively monitor subcutaneous and orthotopic prostate tumor growth and metastasis in vivo. Copyright 2004 Wiley-Liss, Inc.

Effects of AC/DC magnetic fields, frequency, and nanoparticle aspect ratio on cellular transfection of gene vectors

NASA Astrophysics Data System (ADS)

Ford, Kris; Mair, Lamar; Fisher, Mike; Rowshon Alam, Md.; Juliano, Rudolph; Superfine, Richard

2008-10-01

In order to make non-viral gene delivery a useful tool in the study and treatment of genetic disorders, it is imperative that these methodologies be further refined to yield optimal results. Transfection of magnetic nanoparticles and nanorods are used as non-viral gene vectors to transfect HeLa EGFP-654 cells that stably express a mutated enhanced green fluorescent protein (EGFP) gene. We deliver antisense oligonucleotides to these cells designed to correct the aberrant splicing caused by the mutation in the EGFP gene. We also transfect human bronchial endothelial cells and immortalized WI-38 lung cells with pEGFP-N1 vectors. To achieve this we bind the genes to magnetic nanoparticles and nanorods and introduce magnetic fields to effect transfection. We wish to examine the effects of magnetic fields on the transfection of these particles and the benefits of using alternating (AC) magnetic fields in improving transfection rates over direct (DC) magnetic fields. We specifically look at the frequency dependence of the AC field and particle aspect ratio as it pertains to influencing transfection rate. We posit that the increase in angular momentum brought about by the AC field and the high aspect ratio of the nanorod particles, is vital to generating the force needed to move the particle through the cell membrane.

Developing a Predictive Capability for Bioluminescence Signatures

DTIC Science & Technology

2011-09-30

sources of bioluminescence are dinoflagellates, common unicellular plankton that are also known to form red tides. Dinoflagellate bioluminescence is...wakes to locate their prey. The bioluminescence signature of a moving object depends on the bioluminescence potential of the organisms (related to...AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) University of California, San

Bioluminescence.

ERIC Educational Resources Information Center

Jones, M. Gail

1993-01-01

Describes bioluminescence and the chemistry of how it occurs. Presents information for conducting the following classroom activities: (1) firefly mimic; (2) modeling deep-sea fish; (3) sea fireflies; and (4) the chemistry of light. (PR)

Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae

PubMed Central

Krishnamoorthy, Archana

2015-01-01

Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter's signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases. PMID:26162874

Generation of mammalian cells stably expressing multiple genes at predetermined levels.

PubMed

Liu, X; Constantinescu, S N; Sun, Y; Bogan, J S; Hirsch, D; Weinberg, R A; Lodish, H F

2000-04-10

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the

Engineering Bioluminescent Proteins: Expanding their Analytical Potential

PubMed Central

Rowe, Laura; Dikici, Emre; Daunert, Sylvia

2009-01-01

Synopsis Bioluminescence has been observed in nature since the dawn of time, but now, scientists are harnessing it for analytical applications. Laura Rowe, Emre Dikici, and Sylvia Daunert of the University of Kentucky describe the origins of bioluminescent proteins and explore their uses in the modern chemistry laboratory. The cover features spectra of bioluminescent light superimposed on an image of jellyfish, which are a common source of bioluminescent proteins. Images courtesy of Emre Dikici and Shutterstock. PMID:19725502

Bioluminescence Risk Detection Aid

DTIC Science & Technology

2010-01-01

Delivery Vehicle, or diver) bioluminescence, based on local environmental data, in-situ measurements, and simple radiative transfer models. This work...vehicle diving to 5.5 m. Green = REMUS vehicle diving to 6.5 m. Observations were corrected for the angle of observation. IMPACT /APPLICATIONS...will sense vehicle-stimulated bioluminesce, measure local environmental conditions and ingest the information to solve a simple radiative transfer

Bioluminescent organs of two deep-sea arrow worms, Eukrohnia fowleri and Caecosagitta macrocephala, with further observations on Bioluminescence in chaetognaths.

PubMed

Thuesen, Erik V; Goetz, Freya E; Haddock, Steven H D

2010-10-01

Bioluminescence in the deep-sea chaetognath Eukrohnia fowleri is reported for the first time, and behavioral, morphological, and chemical characteristics of bioluminescence in chaetognaths are examined. Until this study, the only known species of bioluminescent chaetognath was Caecosagitta macrocephala. The luminescent organ of that species is located on the ventral edge of each anterior lateral fin, whereas that of E. fowleri runs across the center of the tail fin on both dorsal and ventral sides. Scanning electron microscopy showed that the bioluminescent organs of both species consist of hexagonal chambers containing elongate ovoid particles-the organelles holding bioluminescent materials. No other luminous organism is known to use hexagonal packing to hold bioluminescent materials. Transmission electron microscopy of particles from C. macrocephala revealed a densely packed paracrystalline matrix punctuated by globular inclusions, which likely correspond to luciferin and luciferase, respectively. Both species use unique luciferases in conjunction with coelenterazine for light emission. Luciferase of C. macrocephala becomes inactive after 30 min, but luciferase of E. fowleri is highly stable. Although C. macrocephala has about 90 times fewer particles than E. fowleri, it has a similar bioluminescent capacity (total particle volume) due to its larger particle size. In situ observations of C. macrocephala from a remotely operated vehicle revealed that the luminous particles are released to form a cloud. The discovery of bioluminescence in a second chaetognath phylogenetically distant from the first highlights the importance of bioluminescence among deep-sea organisms.

A Nisin Bioassay Based on Bioluminescence

PubMed Central

Wahlström, G.; Saris, P. E. J.

1999-01-01

A Lactococcus lactis subsp. lactis strain that can sense the bacteriocin nisin and transduce the signal into bioluminescence was constructed. By using this strain, a bioassay based on bioluminescence was developed for quantification of nisin, for detection of nisin in milk, and for identification of nisin-producing strains. As little as 0.0125 ng of nisin per ml was detected within 3 h by this bioluminescence assay. This detection limit was lower than in previously described methods. PMID:10427078

Circadian Control Sheds Light on Fungal Bioluminescence

PubMed Central

Oliveira, Anderson G.; Stevani, Cassius V.; Waldenmaier, Hans E.; Viviani, Vadim; Emerson, Jillian M.; Loros, Jennifer J.; Dunlap, Jay C.

2015-01-01

Summary Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1-5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi – only 71 species, all within the ~9000 fungi of the temperate and tropical Agaricales Order - are reported from among ~100,000 described fungal species [6,7]. All require oxygen [8] and energy (NADH or NADPH) for bioluminescence, and are reported to emit green light (λmax 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a by-product of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and the luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millenia [8-15], we examined interactions between luminescent fungi and insects [16]. Prosthetic acrylic resin “mushrooms”, internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans) as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants) at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy where wind flow is greatly reduced. PMID:25802150

Developing a Predictive Capability for Bioluminescence Signatures

DTIC Science & Technology

2012-09-30

dinoflagellates, common unicellular plankton that are also known to form red tides. Dinoflagellate bioluminescence is stimulated by flow stress of...bioluminescence signature of a moving object depends on the bioluminescence potential of the organisms (related to their species abundance), the...WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Naval Surface Warfare Center Panama City, FL 32407 8. PERFORMING ORGANIZATION

Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

PubMed

Gaji, Rajshekhar Y; Zhang, Deqing; Breathnach, Cormac C; Vaishnava, Shipra; Striepen, Boris; Howe, Daniel K

2006-11-01

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

[Rapid bioluminescent antibiotic susceptibility assay].

PubMed

Frundzhian, V G; Ugarova, N N; Blatun, L A; Terekhova, R P; Rusanova, E V

2009-01-01

Rapid testing of pathogen susceptibility to antibiotics is of great practical value for rational chemotherapy of pyoinflammatory deseases and postoperative complications of microbial etiology. The standard microbiological methods, i.e., the disk diffusion method and the method of serial dilutions are labour- and time-consuming (not less than 18-36 hours). The method of the authors is based on measuring bioluminescence resulting from interaction of adenosine-5'-triphosphate (ATP) and ATP reagent, a standard reaction mixture of firefly luciferase (an enzyme) and luciferin. The bioluminescence intensity is proportional to the ATP concentration in the reaction mixture and the ATP concentration is proportional to the number of the pathogen viable cells in the sample. The bioluminescence intensity value in the pathogen suspension aliquots with and without (control) the antibiotic were compared after the incubation for 5 hours and the coefficient of the microbial cell growth inhibition was calculated. Satisfactory correlation (R2 > 88%) of the results of the bioluminescent assay and the assay with the disk diffusion method and the method of serial dilutions was observed.

Thoughts on the diversity of convergent evolution of bioluminescence on earth

NASA Astrophysics Data System (ADS)

Waldenmaier, Hans E.; Oliveira, Anderson G.; Stevani, Cassius V.

2012-10-01

The widespread independent evolution of analogous bioluminescent systems is one of the most impressive and diverse examples of convergent evolution on earth. There are roughly 30 extant bioluminescent systems that have evolved independently on Earth, with each system likely having unique enzymes responsible for catalysing the bioluminescent reaction. Bioluminescence is a chemical reaction involving a luciferin molecule and a luciferase or photoprotein that results in the emission of light. Some independent systems utilize the same luciferin, such as the use of tetrapyrrolic compounds by krill and dinoflagellates, and the wide use of coelenterazine by marine organisms, while the enzymes involved are unique. One common thread among all the different bioluminescent systems is the requirement of molecular oxygen. Bioluminescence is found in most forms of life, especially marine organisms. Bioluminescence in known to benefit the organism by: attraction, repulsion, communication, camouflage, and illumination. The marine ecosystem is significantly affected by bioluminescence, the only light found in the pelagic zone and below is from bioluminescent organisms. Transgenic bioluminescent organisms have revolutionized molecular research, medicine and the biotechnology industry. The use of bioluminescence in studying molecular pathways and disease allows for non-invasive and real-time analysis. Bioluminescence-based assays have been developed for several analytes by coupling luminescence to many enzyme-catalysed reactions.

Nanostructured biosensor using bioluminescence quenching technique for glucose detection.

PubMed

Chen, Longyan; Chen, Longyi; Dotzert, Michelle; Melling, C W James; Zhang, Jin

2017-08-22

Most methods for monitoring glucose level require an external energy source which may limit their application, particularly in vivo test. Bioluminescence technique offers an alternative way to provide emission light without external energy source by using bioluminescent proteins found from firefly or marine vertebrates and invertebrates. For quick and non-invasive detection of glucose, we herein developed a nanostructured biosensor by applying the bioluminescence technique. Luciferase bioluminescence protein (Rluc) is conjugated with β-cyclodextrin (β-CD). The bioluminescence intensity of Rluc can be quenched by 8 ± 3 nm gold nanoparticles (Au NPs) when Au NPs covalently bind to β-CD. In the presence of glucose, Au NPs are replaced and leave far from Rluc through a competitive reaction, which results in the restored bioluminescence intensity of Rluc. A linear relationship is observed between the restored bioluminescence intensity and the logarithmic glucose concentration in the range of 1-100 µM. In addition, the selectivity of this designed sensor has been evaluated. The performance of the senor for determination of the concentration of glucose in the blood of diabetic rats is studied for comparison with that of the concentration of glucose in aqueous. This study demonstrates the design of a bioluminescence sensor for quickly detecting the concentration of glucose sensitively.

Bioluminescence of Marine Dinoflagellates

PubMed Central

Seliger, H. H.; Fastie, W. G.; Taylor, W. R.; McElroy, W. D.

1962-01-01

Portable light-baffled underwater photometers have been designed for the measurement of dinoflagellate bioluminescence by day and night. Maximal light emission is obtained by mechanical stimulation in a defined volume. The pump which stimulates the dinoflagellates also constantly replenishes the sample volume so that continuous measurements are possible. Evidence for both diurnal variation and vertical migration is presented. Using luminous bacteria for calibration a single dinoflagellate has been found to emit of the order of 1010 light quanta per flash. The technique suggests that large scale mapping of bioluminescence is feasible. PMID:19873546

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokawa, Satoru; School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650; Suzuki, Takahiro

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase frommore » the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion. - Highlights: • The fused protein of proglucagon to Gaussia luciferase was used as a reporter. • The fusion protein was highly expressed using a preferred human-codon optimized gene. • Glucagon secretion stimulated by depolarization was determined by luminescence. • Glucagon secretion in α cells was visualized by bioluminescence imaging. • Glucagon secretion sites were localized in the intercellular contact regions.« less

In vivo bioluminescence imaging using orthotopic xenografts towards patient's derived-xenograft Medulloblastoma models.

PubMed

Asadzadeh, Fatemeh; Ferrucci, Veronica; DE Antonellis, Pasqualino; Zollo, Massimo

2017-03-01

Medulloblastoma is a cerebellar neoplasia of the central nervous system. Four molecular subgrups have been identified (MBWNT, MBSHH, MBgroup3 and MBgroup4) with distinct genetics and clinical outcome. Among these, MBgroup3-4 are highly metastatic with the worst prognosis. The current standard therapy includes surgery, radiation and chemotherapy. Thus, specific treatments adapted to cure those different molecular subgroups are needed. The use of orthotopic xenograft models, together with the non-invasive in vivo biolumiscence imaging (BLI) technology, is emerging during preclinical studies to test novel therapeutics for medulloblastoma treatment. Orthotopic MB xenografts were performed by injection of Daoy-luc cells, that had been previously infected with lentiviral particles to stably express luciferase gene, into the fourth right ventricle of the cerebellum of ten nude mice. For the implantation, specific stereotactic coordinates were used. Seven days after the implantation the mice were imaged by acquisitions of bioluminescence imaging (BLI) using IVIS 3D Illumina Imaging System (Xenogen). Tumor growth was evaluated by quantifying the bioluminescence signals using the integrated fluxes of photons within each area of interest using the Living Images Software Package 3.2 (Xenogen-Perkin Elmer). Finally, histological analysis using hematoxylin-eosin staining was performed to confirm the presence of tumorigenic cells into the cerebellum of the mice. We describe a method to use the in vivo bioluminescent imaging (BLI) showing the potential to be used to investigate the potential antitumorigenic effects of a drug for in vivo medulloblastoma treatment. We also discuss other studies in which this technology has been applied to obtain a more comprehensive knowledge of medulloblastoma using orthotopic xenograft mouse models. There is a need to develop patient's derived-xenograft (PDX) model systems to test novel drugs for medulloblastoma treatment within each molecular sub

Intracranial implantation with subsequent 3D in vivo bioluminescent imaging of murine gliomas.

PubMed

Abdelwahab, Mohammed G; Sankar, Tejas; Preul, Mark C; Scheck, Adrienne C

2011-11-06

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain (1); therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation (1). Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1 mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4 mm to a depth of 2.6 mm. Two μl of GL261-luc or GL261-luc2 cells (10(7) cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured. Following stereotactic implantation the bioluminescent cells are

Interactive graphic editing tools in bioluminescent imaging simulation

NASA Astrophysics Data System (ADS)

Li, Hui; Tian, Jie; Luo, Jie; Wang, Ge; Cong, Wenxiang

2005-04-01

It is a challenging task to accurately describe complicated biological tissues and bioluminescent sources in bioluminescent imaging simulation. Several graphic editing tools have been developed to efficiently model each part of the bioluminescent simulation environment and to interactively correct or improve the initial models of anatomical structures or bioluminescent sources. There are two major types of graphic editing tools: non-interactive tools and interactive tools. Geometric building blocks (i.e. regular geometric graphics and superquadrics) are applied as non-interactive tools. To a certain extent, complicated anatomical structures and bioluminescent sources can be approximately modeled by combining a sufficient large number of geometric building blocks with Boolean operators. However, those models are too simple to describe the local features and fine changes in 2D/3D irregular contours. Therefore, interactive graphic editing tools have been developed to facilitate the local modifications of any initial surface model. With initial models composed of geometric building blocks, interactive spline mode is applied to conveniently perform dragging and compressing operations on 2D/3D local surface of biological tissues and bioluminescent sources inside the region/volume of interest. Several applications of the interactive graphic editing tools will be presented in this article.

Vision and Bioluminescence in the Deep-sea Benthos

NASA Astrophysics Data System (ADS)

Frank, T. M.; Johnsen, S.; Bracken-Grissom, H.; Messing, C. G.; Widder, E.

2016-02-01

During a NOAA-OER funded research cruise, novel collecting techniques were used to collect live, deep-sea benthic animals for studies of bioluminescence and vision. True color images and emission spectra of bioluminescence were obtained from a number of species, including the spiral octocoral Iridogorgia sp., the sea fan Chrysogorgia sp., the sea pen Umbellula sp., and the caridean shrimp Heterocarpus oryx. Electrophysiological studies were conducted on 3 species of decapod crustaceans collected with methods that limited light damage to their photoreceptors. The caridean shrimp, Bathypalaemonella, collected from 1920 m, was always found in association with the bioluminescent spiral octocoral Iridogorgia. While moribund at the surface, enough data were obtained from one specimen to show different waveforms in response to short and long wavelength light, indicative of two different classes of photoreceptor cells. The chirostylid crab, Uroptychus nitidus, found in association with the bioluminescent sea fan, Chrysogorgia sp., also appears to possess two visual pigments, and if further analysis of data supports this preliminary observation, will be the 4th species of deep-sea, non-bioluminescent crustaceans possessing two visual pigments found in association with bioluminescent cnidarians. These four species also share another characteristic - the presence of one or two very long claws, which the crab species are known to use to pick items (possibly plankton stuck in the mucus) off their cnidarian hosts. These data support the previously presented hypothesis (Frank et al. 2012), that these crustaceans may be utilizing their dual visual pigment systems to distinguish between prey and host, based on spectral differences between pelagic and benthic bioluminescence.

Regulation of early gene expression from the bovine papillomavirus genome in transiently transfected C127 cells.

PubMed Central

Szymanski, P; Stenlund, A

1991-01-01

Expression of bovine papillomavirus (BPV) early gene products is required for viral DNA replication and establishment of the transformed phenotype. By the use of a highly efficient electroporation system, we have examined for the first time the transcriptional activity of BPV promoters in their natural genomic context in a replication-permissive cell line. We have determined that a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. This suggests that the transcriptional activity of the BPV genome in stably transformed cells represents the early stage of BPV gene expression. Quantitative differences in promoter activity between transiently transfected and stably transformed cells suggest that subtle changes in gene expression may control progression of the viral life cycle. Deletion analysis demonstrated that the E2 transactivator protein stimulates all of the early promoters through sequences located in the upstream regulatory region. This E2-dependent enhancer was found to be highly redundant, and particular E2 binding sites did not display a preference for particular promoters. Despite this dependence on a common cis-acting sequence, the various promoters displayed different sensitivities to the E2 transactivator. The findings that E2 regulates all promoters and, with the exception of the E2 repressors, that no other known viral gene product appears to affect transcription indicate that the E2 system functions as the master regulator of BPV early gene expression. Images PMID:1656065

Bioluminescence as an ecological factor during high Arctic polar night

NASA Astrophysics Data System (ADS)

Cronin, Heather A.; Cohen, Jonathan H.; Berge, Jørgen; Johnsen, Geir; Moline, Mark A.

2016-11-01

Bioluminescence commonly influences pelagic trophic interactions at mesopelagic depths. Here we characterize a vertical gradient in structure of a generally low species diversity bioluminescent community at shallower epipelagic depths during the polar night period in a high Arctic fjord with in situ bathyphotometric sampling. Bioluminescence potential of the community increased with depth to a peak at 80 m. Community composition changed over this range, with an ecotone at 20-40 m where a dinoflagellate-dominated community transitioned to dominance by the copepod Metridia longa. Coincident at this depth was bioluminescence exceeding atmospheric light in the ambient pelagic photon budget, which we term the bioluminescence compensation depth. Collectively, we show a winter bioluminescent community in the high Arctic with vertical structure linked to attenuation of atmospheric light, which has the potential to influence pelagic ecology during the light-limited polar night.

Bioluminescent bioreporter sensing of foodborne toxins

NASA Astrophysics Data System (ADS)

Fraley, Amanda C.; Ripp, Steven; Sayler, Gary S.

2004-06-01

Histamine is the primary etiological agent in the foodborne disease scombrotoxicosis, one of the most common food toxicities related to fish consumption. Procedures for detecting histamine in fish products are available, but are often too expensive or too complex for routine use. As an alternative, a bacterial bioluminescent bioreporter has been constructed to develop a biosensor system that autonomously responds to low levels of histamine. The bioreporter contains a promoterless Photorhabdus luminescens lux operon (luxCDABE) fused with the Vibrio anguillarum angR regulatory gene promoter of the anguibactin biosynthetic operon. The bioreporter emitted 1.46 times more bioluminescence than background, 30 minutes after the addition of 100mM histamine. However, specificity was not optimal, as this biosensor generated significant bioluminescence in the presence of L-proline and L-histidine. As a means towards improving histamine specificity, the promoter region of a histamine oxidase gene from Arthrobacter globiformis was cloned upstream of the promotorless lux operon from Photorhabdus luminescens. This recently constructed whole-cell, lux-based bioluminescent bioreporter is currently being tested for optimal performance in the presence of histamine in order to provide a rapid, simple, and inexpensive model sensor for the detection of foodborne toxins.

Measurement of Bacterial Bioluminescence Intensity and Spectrum: Current Physical Techniques and Principles.

PubMed

Jia, Kun; Ionescu, Rodica Elena

2016-01-01

: Bioluminescence is light production by living organisms, which can be observed in numerous marine creatures and some terrestrial invertebrates. More specifically, bacterial bioluminescence is the "cold light" produced and emitted by bacterial cells, including both wild-type luminescent and genetically engineered bacteria. Because of the lively interplay of synthetic biology, microbiology, toxicology, and biophysics, different configurations of whole-cell biosensors based on bacterial bioluminescence have been designed and are widely used in different fields, such as ecotoxicology, food toxicity, and environmental pollution. This chapter first discusses the background of the bioluminescence phenomenon in terms of optical spectrum. Platforms for bacterial bioluminescence detection using various techniques are then introduced, such as a photomultiplier tube, charge-coupled device (CCD) camera, micro-electro-mechanical systems (MEMS), and complementary metal-oxide-semiconductor (CMOS) based integrated circuit. Furthermore, some typical biochemical methods to optimize the analytical performances of bacterial bioluminescent biosensors/assays are reviewed, followed by a presentation of author's recent work concerning the improved sensitivity of a bioluminescent assay for pesticides. Finally, bacterial bioluminescence as implemented in eukaryotic cells, bioluminescent imaging, and cancer cell therapies is discussed.

A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system.

PubMed

Zhang, Li; Li, Qianqian; Liu, Qiang; Huang, Weijin; Nie, Jianhui; Wang, Youchun

2017-08-03

Marburg virus (MARV) can cause lethal hemorrhagic fever in humans. Handling of MARV is restricted to high-containment biosafety level 4 (BSL-4) facilities, which greatly impedes research into this virus. In this study, a high titer of MARV pseudovirus was generated through optimization of the HIV backbone vectors, the ratio of backbone vector to MARV glycoprotein expression vector, and the transfection reagents. An in vitro neutralization assay and an in vivo bioluminescent imaging mouse model for MARV were developed based on the pseudovirus. Protective serum against MARV was successfully induced in guinea pigs, which showed high neutralization activity in vitro and could also protect Balb/c mice from MARV pseudovirus infection in vivo. This system could be a convenient tool to enable the evaluation of vaccines and therapeutic drugs against MARV in non-BSL-4 laboratories.

Retinoblastoma protein (pRB) was significantly phosphorylated through a Ras-to-MAPK pathway in mutant K-ras stably transfected human adrenocortical cells.

PubMed

Chen, Y-F; Chiu, H-H; Wu, C-H; Wang, J-Y; Chen, F-M; Tzou, W-H; Shin, S-J; Lin, S-R

2003-10-01

Our previous studies have shown that the cell proliferation rate, mRNA levels of p450scc, p450c17, and 3betaHSD, and secretion of cortisol were significantly increased in human adrenocortical cells stably transfected with mutated K-ras expression plasmid "pK568MRSV" after being inducted with IPTG. In addition, the increased level was a time-dependent manner. However, the levels of p450, p450scc, p450c17, 3betaHSD, cortisol, and cell proliferation rate were inhibited by a MEK phospholation inhibitor, PD098059. The above results prove that mutated K-ras oncogene is able to regulate tumorigenesis and steroidogenesis through a Ras-RAF-MEK-MAPK signal transduction pathway. The aim of this study was to investigate regulated factors in this pathway and also examine whether the other signal transduction pathways or other moles involved in tumorigenesis or steroidogenesis. In the first year, we analyzed gene profiles of mutant K-ras-transfected adrenocortical cells by DNA microarray to determine the gene expression related to cell cycle, signal transduction, apoptosis, tumorigenesis, steroidogenesis, and other expressed sequence tag. After being affected by the K-ras mutant, gene expression was significantly increased in some upregulated genes. Human zinc-finger protein 22 increased by 28.5 times, Osteopontin increased by 5.8 times, LIM domain Kinase 2 (LIMK2) increased by 3.3 times, Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated Kinase 2 (DYRK2) increased by 2.2 times, and human syntaxin 3 increased by two times. On the other hand, significant decreases in gene expression were also observed in some downregulated genes. Retinoblastoma binding protein 1 (RBBP1) decreased by four times, Homo sapiens craniofacial development protein 1 (CFDP1) decreased by 2.4 times, DAP Kinase-related apoptosis-inducing protein Kinase 1 (DRAK1) decreased by 2.3 times, SKI-interacting protein (SKIP) decreased by 2.2 times, and human poly(A)-Binding protein (PABP) decreased

A REVIEW OF ENVIRONMENTAL APPLICATIONS OF BIOLUMINESCENCE MEASUREMENTS

EPA Science Inventory

This review of the recent literature on environmental applications of bioluminescence systems will focus on in vivo and in vitro bioluminescence methods that have been utilized to elucidate properties of chemicals, toxic and mutagenic effects, and to estimate biomass. The unifyin...

Transfection using DEAE-dextran.

PubMed

Gulick, T

2001-05-01

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

Transfection using DEAE-dextran.

PubMed

Gulick, Tod

2003-08-01

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

Transfection using DEAE-dextran.

PubMed

Gulick, T

2001-05-01

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)-dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE-dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE-dextran transfection, as well as two more specific protocols for typical experimental applications. The Basic Protocol is suitable for transfection of anchorage-dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE-dextran-mediated transfection can be used.

Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents

PubMed Central

Hunt, Michelle A.; Currie, Margaret J.; Robinson, Bridget A.; Dachs, Gabi U.

2010-01-01

Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfection, followed by flow cytometry of transfected HUVEC to determine the proportion of EGFP-expressing cells as a measure of transfection efficiency. Lipofectamine 2000 and Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) gave the highest transfection efficiencies of the reagents tested. Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays. PMID:20592869

Detection of ATP and NADH: A Bioluminescent Experience.

ERIC Educational Resources Information Center

Selig, Ted C.; And Others

1984-01-01

Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

Bioluminescence-Activated Deep-Tissue Photodynamic Therapy of Cancer

PubMed Central

Kim, Yi Rang; Kim, Seonghoon; Choi, Jin Woo; Choi, Sung Yong; Lee, Sang-Hee; Kim, Homin; Hahn, Sei Kwang; Koh, Gou Young; Yun, Seok Hyun

2015-01-01

Optical energy can trigger a variety of photochemical processes useful for therapies. Owing to the shallow penetration of light in tissues, however, the clinical applications of light-activated therapies have been limited. Bioluminescence resonant energy transfer (BRET) may provide a new way of inducing photochemical activation. Here, we show that efficient bioluminescence energy-induced photodynamic therapy (PDT) of macroscopic tumors and metastases in deep tissue. For monolayer cell culture in vitro incubated with Chlorin e6, BRET energy of about 1 nJ per cell generated as strong cytotoxicity as red laser light irradiation at 2.2 mW/cm2 for 180 s. Regional delivery of bioluminescence agents via draining lymphatic vessels killed tumor cells spread to the sentinel and secondary lymph nodes, reduced distant metastases in the lung and improved animal survival. Our results show the promising potential of novel bioluminescence-activated PDT. PMID:26000054

Bioluminescence lights the way to food safety

NASA Astrophysics Data System (ADS)

Brovko, Lubov Y.; Griffiths, Mansel W.

2003-07-01

The food industry is increasingly adopting food safety and quality management systems that are more proactive and preventive than those used in the past which have tended to rely on end product testing and visual inspection. The regulatory agencies in many countries are promoting one such management tool, Hazard Analysis Critical Control Point (HACCP), as a way to achieve a safer food supply and as a basis for harmonization of trading standards. Verification that the process is safe must involve microbiological testing but the results need not be generated in real-time. Of all the rapid microbiological tests currently available, the only ones that come close to offering real-time results are bioluminescence-based methods. Recent developments in application of bioluminescence for food safety issues are presented in the paper. These include the use of genetically engineered microorganisms with bioluminescent and fluorescent phenotypes as a real time indicator of physiological state and survival of food-borne pathogens in food and food processing environments as well as novel bioluminescent-based methods for rapid detection of pathogens in food and environmental samples. Advantages and pitfalls of the methods are discussed.

In Vivo Bioluminescence Imaging for Longitudinal Monitoring of Inflammation in Animal Models of Uveitis.

PubMed

Gutowski, Michal B; Wilson, Leslie; Van Gelder, Russell N; Pepple, Kathryn L

2017-03-01

We develop a quantitative bioluminescence assay for in vivo longitudinal monitoring of inflammation in animal models of uveitis. Three models of experimental uveitis were induced in C57BL/6 albino mice: primed mycobacterial uveitis (PMU), endotoxin-induced uveitis (EIU), and experimental autoimmune uveitis (EAU). Intraperitoneal injection of luminol sodium salt, which emits light when oxidized, provided the bioluminescence substrate. Bioluminescence images were captured by a PerkinElmer In Vivo Imaging System (IVIS) Spectrum and total bioluminescence was analyzed using Living Image software. Bioluminescence on day zero was compared to bioluminescence on the day of peak inflammation for each model. Longitudinal bioluminescence imaging was performed in EIU and EAU. In the presence of luminol, intraocular inflammation generates detectable bioluminescence in three mouse models of uveitis. Peak bioluminescence in inflamed PMU eyes (1.46 × 105 photons/second [p/s]) was significantly increased over baseline (1.47 × 104 p/s, P = 0.01). Peak bioluminescence in inflamed EIU eyes (3.18 × 104 p/s) also was significantly increased over baseline (1.09 × 104 p/s, P = 0.04), and returned to near baseline levels by 48 hours. In EAU, there was a nonsignificant increase in bioluminescence at peak inflammation. In vivo bioluminescence may be used as a noninvasive, quantitative measure of intraocular inflammation in animal models of uveitis. Primed mycobacterial uveitis and EIU are both acute models with robust anterior inflammation and demonstrated significant changes in bioluminescence corresponding with peak inflammation. Experimental autoimmune uveitis is a more indolent posterior uveitis and generated a more modest bioluminescent signal. In vivo imaging system bioluminescence is a nonlethal, quantifiable assay that can be used for monitoring inflammation in animal models of uveitis.

Bioluminescent Reaction by Immobilized Luciferase

NASA Astrophysics Data System (ADS)

Tanaka, Ryuta; Takahama, Eriko; Iinuma, Masataka; Ikeda, Takeshi; Kadoya, Yutaka; Kuroda, Akio

We have investigated an effect of immobilization of luciferase molecules at the optical fiber end on a bioluminescent reaction. The time dependence of measured count rates of emitted photons has been analyzed by fitting with numerical solution of differential equations including the effect of the product-inhibitor and the deactivation of the luciferase. Through the analysis, we have successfully extracted kinetic constants such as, reaction rate, number of active luciferase molecules, etc. Ratio of active molecules to total luciferase molecules in immobilization was one order of magnitude lower than that in solution. The reaction rate of the bioluminescent process was also different from the one of free luciferase in solution.

Bioluminescence in the Ocean: Origins of Biological, Chemical, and Ecological Diversity

NASA Astrophysics Data System (ADS)

Widder, E. A.

2010-05-01

From bacteria to fish, a remarkable variety of marine life depends on bioluminescence (the chemical generation of light) for finding food, attracting mates, and evading predators. Disparate biochemical systems and diverse phylogenetic distribution patterns of light-emitting organisms highlight the ecological benefits of bioluminescence, with biochemical and genetic analyses providing new insights into the mechanisms of its evolution. The origins and functions of some bioluminescent systems, however, remain obscure. Here, I review recent advances in understanding bioluminescence in the ocean and highlight future research efforts that will unite molecular details with ecological and evolutionary relationships.

Bioluminescence Monitoring of Neuronal Activity in Freely Moving Zebrafish Larvae

PubMed Central

Knafo, Steven; Prendergast, Andrew; Thouvenin, Olivier; Figueiredo, Sophie Nunes; Wyart, Claire

2017-01-01

The proof of concept for bioluminescence monitoring of neural activity in zebrafish with the genetically encoded calcium indicator GFP-aequorin has been previously described (Naumann et al., 2010) but challenges remain. First, bioluminescence signals originating from a single muscle fiber can constitute a major pitfall. Second, bioluminescence signals emanating from neurons only are very small. To improve signals while verifying specificity, we provide an optimized 4 steps protocol achieving: 1) selective expression of a zebrafish codon-optimized GFP-aequorin, 2) efficient soaking of larvae in GFP-aequorin substrate coelenterazine, 3) bioluminescence monitoring of neural activity from motor neurons in free-tailed moving animals performing acoustic escapes and 4) verification of the absence of muscle expression using immunohistochemistry. PMID:29130058

Transfection using DEAE-dextran.

PubMed

Selden, R F

2001-05-01

Two protocols for DEAE-dextran transfection of cells are provided in this unit. The Basic Protocol describes a procedure used to transfect adherent cells and the first Alternate Protocol presents a method used to transfect suspension cells. If an increase in transfection efficiency is needed, cells can be treated with chloroquine as described in the second Alternate Protocol.

Advances in bioluminescence imaging: new probes from old recipes.

PubMed

Yao, Zi; Zhang, Brendan S; Prescher, Jennifer A

2018-06-04

Bioluminescent probes are powerful tools for visualizing biology in live tissues and whole animals. Recent years have seen a surge in the number of new luciferases, luciferins, and related tools available for bioluminescence imaging. Many were crafted using classic methods of optical probe design and engineering. Here we highlight recent advances in bioluminescent tool discovery and development, along with applications of the probes in cells, tissues, and organisms. Collectively, these tools are improving in vivo imaging capabilities and bolstering new research directions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering

PubMed Central

Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies. PMID:26407291

Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering.

PubMed

Ker, Dai Fei Elmer; Sharma, Rashmi; Wang, Evelyna Tsi Hsin; Yang, Yunzhi Peter

2015-01-01

Mouse C3H10T1/2 fibroblasts are multipotent, mesenchymal stem cell (MSC)-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1/2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1/2 cells and mRuby2-transfected C3H10T1/2 cells stained positive for alkaline phosphatase (ALP) as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1/2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1/2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1/2 cells. These cells will be available from American Type Culture Collection (ATCC; CRL-3268™) and may be a valuable tool for preclinical studies.

Basic and Applied Aspects of Color Tuning of Bioluminescence Systems

NASA Astrophysics Data System (ADS)

Ohmiya, Yoshihiro

2005-09-01

V. Viviani et al. [Biochemistry 38 (1999) 8271] were the first to succeed in cloning the red-emitting enzyme from the South American railroad worm, which is the only bioluminescent organism known to emit a red-colored light. The application of red bioluminescence has been our goal because the transmittance of longer-wavelength light is superior to that of the other colors for visualization of biological functions in living cells. Now, different color luciferases, which emit with wavelength maxima ranging from 400 to 630 nm, are available and are being used. For example, based on different color luciferases, Nakajima et al. developed a tricolor reporter in vitro assay system based on these different color luciferases in which the expression of three genes can be monitored simultaneously. On the other hand, bioluminescence resonance energy transfer (BRET) is a natural phenomenon caused by the intermolecular interaction between a bioluminescent protein and a fluorophore on a second protein, resulting in the light from the bioluminescence reaction having the spectrum of the fluorophore. Otsuji et al. [Anal. Biochem. 329 (2004) 230] showed that the change in the efficiency of energy transfer in intramolecular BRET can quantify cellular functions in living cells. In this review, I introduce the basic mechanisms of color tuning in bioluminescent systems and new applications based on color tuning in the life sciences.

Imaging of Bubonic Plague Dynamics by In Vivo Tracking of Bioluminescent Yersinia pestis

PubMed Central

Nham, Toan; Filali, Sofia; Danne, Camille; Derbise, Anne; Carniel, Elisabeth

2012-01-01

Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response. PMID:22496846

Imaging of bubonic plague dynamics by in vivo tracking of bioluminescent Yersinia pestis.

PubMed

Nham, Toan; Filali, Sofia; Danne, Camille; Derbise, Anne; Carniel, Elisabeth

2012-01-01

Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.

A gantry-based tri-modality system for bioluminescence tomography

PubMed Central

Yan, Han; Lin, Yuting; Barber, William C.; Unlu, Mehmet Burcin; Gulsen, Gultekin

2012-01-01

A gantry-based tri-modality system that combines bioluminescence (BLT), diffuse optical (DOT), and x-ray computed tomography (XCT) into the same setting is presented here. The purpose of this system is to perform bioluminescence tomography using a multi-modality imaging approach. As parts of this hybrid system, XCT and DOT provide anatomical information and background optical property maps. This structural and functional a priori information is used to guide and restrain bioluminescence reconstruction algorithm and ultimately improve the BLT results. The performance of the combined system is evaluated using multi-modality phantoms. In particular, a cylindrical heterogeneous multi-modality phantom that contains regions with higher optical absorption and x-ray attenuation is constructed. We showed that a 1.5 mm diameter bioluminescence inclusion can be localized accurately with the functional a priori information while its source strength can be recovered more accurately using both structural and the functional a priori information. PMID:22559540

A Causal Relation between Bioluminescence and Oxygen to Quantify the Cell Niche

PubMed Central

Lambrechts, Dennis; Roeffaers, Maarten; Goossens, Karel; Hofkens, Johan; Van de Putte, Tom; Schrooten, Jan; Van Oosterwyck, Hans

2014-01-01

Bioluminescence imaging assays have become a widely integrated technique to quantify effectiveness of cell-based therapies by monitoring fate and survival of transplanted cells. To date these assays are still largely qualitative and often erroneous due to the complexity and dynamics of local micro-environments (niches) in which the cells reside. Here, we report, using a combined experimental and computational approach, on oxygen that besides being a critical niche component responsible for cellular energy metabolism and cell-fate commitment, also serves a primary role in regulating bioluminescent light kinetics. We demonstrate the potential of an oxygen dependent Michaelis-Menten relation in quantifying intrinsic bioluminescence intensities by resolving cell-associated oxygen gradients from bioluminescent light that is emitted from three-dimensional (3D) cell-seeded hydrogels. Furthermore, the experimental and computational data indicate a strong causal relation of oxygen concentration with emitted bioluminescence intensities. Altogether our approach demonstrates the importance of oxygen to evolve towards quantitative bioluminescence and holds great potential for future microscale measurement of oxygen tension in an easily accessible manner. PMID:24840204

A Real-Time Non-invasive Auto-bioluminescent Urinary Bladder Cancer Xenograft Model.

PubMed

John, Bincy Anu; Xu, Tingting; Ripp, Steven; Wang, Hwa-Chain Robert

2017-02-01

The study was to develop an auto-bioluminescent urinary bladder cancer (UBC) xenograft animal model for pre-clinical research. The study used a humanized, bacteria-originated lux reporter system consisting of six (luxCDABEfrp) genes to express components required for producing bioluminescent signals in human UBC J82, J82-Ras, and SW780 cells without exogenous substrates. Immune-deficient nude mice were inoculated with Lux-expressing UBC cells to develop auto-bioluminescent xenograft tumors that were monitored by imaging and physical examination. Lux-expressing auto-bioluminescent J82-Lux, J82-Ras-Lux, and SW780-Lux cell lines were established. Xenograft tumors derived from tumorigenic Lux-expressing auto-bioluminescent J82-Ras-Lux cells allowed a serial, non-invasive, real-time monitoring by imaging of tumor development prior to the presence of palpable tumors in animals. Using Lux-expressing auto-bioluminescent tumorigenic cells enabled us to monitor the entire course of xenograft tumor development through tumor cell implantation, adaptation, and growth to visible/palpable tumors in animals.

A causal relation between bioluminescence and oxygen to quantify the cell niche.

PubMed

Lambrechts, Dennis; Roeffaers, Maarten; Goossens, Karel; Hofkens, Johan; Vande Velde, Greetje; Van de Putte, Tom; Schrooten, Jan; Van Oosterwyck, Hans

2014-01-01

Bioluminescence imaging assays have become a widely integrated technique to quantify effectiveness of cell-based therapies by monitoring fate and survival of transplanted cells. To date these assays are still largely qualitative and often erroneous due to the complexity and dynamics of local micro-environments (niches) in which the cells reside. Here, we report, using a combined experimental and computational approach, on oxygen that besides being a critical niche component responsible for cellular energy metabolism and cell-fate commitment, also serves a primary role in regulating bioluminescent light kinetics. We demonstrate the potential of an oxygen dependent Michaelis-Menten relation in quantifying intrinsic bioluminescence intensities by resolving cell-associated oxygen gradients from bioluminescent light that is emitted from three-dimensional (3D) cell-seeded hydrogels. Furthermore, the experimental and computational data indicate a strong causal relation of oxygen concentration with emitted bioluminescence intensities. Altogether our approach demonstrates the importance of oxygen to evolve towards quantitative bioluminescence and holds great potential for future microscale measurement of oxygen tension in an easily accessible manner.

Increased bioassay sensitivity of bioactive molecule discovery using metal-enhanced bioluminescence

NASA Astrophysics Data System (ADS)

Golberg, Karina; Elbaz, Amit; McNeil, Ronald; Kushmaro, Ariel; Geddes, Chris D.; Marks, Robert S.

2014-12-01

We report the use of bioluminescence signal enhancement via proximity to deposited silver nanoparticles for bioactive compound discovery. This approach employs a whole-cell bioreporter harboring a plasmid-borne fusion of a specific promoter incorporated with a bioluminescence reporter gene. The silver deposition process was first optimized to provide optimal nanoparticle size in the reaction time dependence with fluorescein. The use of silver deposition of 350 nm particles enabled the doubling of the bioluminescent signal amplitude by the bacterial bioreporter when compared to an untouched non-silver-deposited microtiter plate surface. This recording is carried out in the less optimal but necessary far-field distance. SEM micrographs provided a visualization of the proximity of the bioreporter to the silver nanoparticles. The electromagnetic field distributions around the nanoparticles were simulated using Finite Difference Time Domain, further suggesting a re-excitation of non-chemically excited bioluminescence in addition to metal-enhanced bioluminescence. The possibility of an antiseptic silver effect caused by such a close proximity was eliminated disregarded by the dynamic growth curves of the bioreporter strains as seen using viability staining. As a highly attractive biotechnology tool, this silver deposition technique, coupled with whole-cell sensing, enables increased bioluminescence sensitivity, making it especially useful for cases in which reporter luminescence signals are very weak.

New bioreactor for in situ simultaneous measurement of bioluminescence and cell density

NASA Astrophysics Data System (ADS)

Picart, Pascal; Bendriaa, Loubna; Daniel, Philippe; Horry, Habib; Durand, Marie-José; Jouvanneau, Laurent; Thouand, Gérald

2004-03-01

This article presents a new device devoted to the simultaneous measurement of bioluminescence and optical density of a bioluminescent bacterial culture. It features an optoelectronic bioreactor with a fully autoclavable module, in which the bioluminescent bacteria are cultivated, a modulated laser diode dedicated to optical density measurement, and a detection head for the acquisition of both bioluminescence and optical density signals. Light is detected through a bifurcated fiber bundle. This setup allows the simultaneous estimation of the bioluminescence and the cell density of the culture medium without any sampling. The bioluminescence is measured through a highly sensitive photomultiplier unit which has been photometrically calibrated to allow light flux measurements. This was achieved by considering the bioluminescence spectrum and the full optical transmission of the device. The instrument makes it possible to measure a very weak light flux of only a few pW. The optical density is determined through the laser diode and a photodiode using numerical synchronous detection which is based on the power spectrum density of the recorded signal. The detection was calibrated to measure optical density up to 2.5. The device was validated using the Vibrio fischeri bacterium which was cultivated under continuous culture conditions. A very good correlation between manual and automatic measurements processed with this instrument has been demonstrated. Furthermore, the optoelectronic bioreactor enables determination of the luminance of the bioluminescent bacteria which is estimated to be 6×10-5 W sr-1 m-2 for optical density=0.3. Experimental results are presented and discussed.

Bioluminescence of beetle luciferases with 6'-amino-D-luciferin analogues reveals excited keto-oxyluciferin as the emitter and phenolate/luciferin binding site interactions modulate bioluminescence colors.

PubMed

Viviani, Vadim R; Neves, Deimison Rodrigues; Amaral, Danilo Trabuco; Prado, Rogilene A; Matsuhashi, Takuto; Hirano, Takashi

2014-08-19

Beetle luciferases produce different bioluminescence colors from green to red using the same d-luciferin substrate. Despite many studies of the mechanisms and structural determinants of bioluminescence colors with firefly luciferases, the identity of the emitters and the specific active site interactions responsible for bioluminescence color modulation remain elusive. To address these questions, we analyzed the bioluminescence spectra with 6'-amino-D-luciferin (aminoluciferin) and its 5,5-dimethyl analogue using a set of recombinant beetle luciferases that naturally elicit different colors and different pH sensitivities (pH-sensitive, Amydetes vivianii λmax=538 nm, Macrolampis sp2 λmax=564 nm; pH-insensitive, Phrixotrix hirtus λmax=623 nm, Phrixotrix vivianii λmax=546 nm, and Pyrearinus termitilluminans λmax=534 nm), a luciferase-like enzyme (Tenebrionidae, Zophobas morio λmax=613 nm), and mutants of C311 (S314). The green-yellow-emitting luciferases display red-shifted bioluminescence spectra with aminoluciferin in relation to those with D-luciferin, whereas the red-emitting luciferases displayed blue-shifted spectra. Bioluminescence spectra with 5,5-dimethylaminoluciferin, in which enolization is blocked, were almost identical to those of aminoluciferin. Fluorescence probing using 2-(4-toluidino)naphthalene-6-sulfonate and inference with aminoluciferin confirm that the luciferin binding site of the red-shifted luciferases is more polar than in the case of the green-yellow-emitting luciferases. Altogether, the results show that the keto form of excited oxyluciferin is the emitter in beetle bioluminescence and that bioluminescence colors are essentially modulated by interactions of the 6'-hydroxy group of oxyluciferin and basic moieties under the influence of the microenvironment polarity of the active site: a strong interaction between a base moiety and oxyluciferin phenol in a hydrophobic microenvironment promotes green-yellow emission, whereas a more polar

Semi-automated Image Processing for Preclinical Bioluminescent Imaging.

PubMed

Slavine, Nikolai V; McColl, Roderick W

Bioluminescent imaging is a valuable noninvasive technique for investigating tumor dynamics and specific biological molecular events in living animals to better understand the effects of human disease in animal models. The purpose of this study was to develop and test a strategy behind automated methods for bioluminescence image processing from the data acquisition to obtaining 3D images. In order to optimize this procedure a semi-automated image processing approach with multi-modality image handling environment was developed. To identify a bioluminescent source location and strength we used the light flux detected on the surface of the imaged object by CCD cameras. For phantom calibration tests and object surface reconstruction we used MLEM algorithm. For internal bioluminescent sources we used the diffusion approximation with balancing the internal and external intensities on the boundary of the media and then determined an initial order approximation for the photon fluence we subsequently applied a novel iterative deconvolution method to obtain the final reconstruction result. We find that the reconstruction techniques successfully used the depth-dependent light transport approach and semi-automated image processing to provide a realistic 3D model of the lung tumor. Our image processing software can optimize and decrease the time of the volumetric imaging and quantitative assessment. The data obtained from light phantom and lung mouse tumor images demonstrate the utility of the image reconstruction algorithms and semi-automated approach for bioluminescent image processing procedure. We suggest that the developed image processing approach can be applied to preclinical imaging studies: characteristics of tumor growth, identify metastases, and potentially determine the effectiveness of cancer treatment.

An Efficient Method for High-Fidelity BAC/PAC Retrofitting with a Selectable Marker for Mammalian Cell Transfection

PubMed Central

Wang, Zunde; Engler, Peter; Longacre, Angelika; Storb, Ursula

2001-01-01

Large-scale genomic sequencing projects have provided DNA sequence information for many genes, but the biological functions for most of them will only be known through functional studies. Bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) are large genomic clones stably maintained in bacteria and are very important in functional studies through transfection because of their large size and stability. Because most BAC or PAC vectors do not have a mammalian selection marker, transfecting mammalian cells with genes cloned in BACs or PACs requires the insertion into the BAC/PAC of a mammalian selectable marker. However, currently available procedures are not satisfactory in efficiency and fidelity. We describe a very simple and efficient procedure that allows one to retrofit dozens of BACs in a day with no detectable deletions or unwanted recombination. We use a BAC/PAC retrofitting vector that, on transformation into competent BAC or PAC strains, will catalyze the specific insertion of itself into BAC/PAC vectors through in vivo cre/loxP site-specific recombination. PMID:11156622

Glowing Worms: Biological, Chemical, and Functional Diversity of Bioluminescent Annelids.

PubMed

Verdes, Aida; Gruber, David F

2017-07-01

Bioluminescence, the ability to produce light by living organisms, has evolved independently in numerous lineages across the tree of life. Luminous forms are found in a wide range of taxonomic groups from bacteria to vertebrates, although the great majority of bioluminescent organisms are marine taxa. Within the phylum Annelida, bioluminescence is widespread, present in at least 98 terrestrial and marine species that represent 45 genera distributed in thirteen lineages of clitellates and polychaetes. The ecological diversity of luminous annelids is unparalleled, with species occupying a great variety of habitats including both terrestrial and marine ecosystems, from coastal waters to the deep-sea, in benthic and pelagic habitats from polar to tropical regions. This great taxonomic and ecological diversity is matched by the wide array of bioluminescent colors-including yellow light, which is very rare among marine taxa-different emission wavelengths even between species of the same genus, and varying patterns, chemical reactions and kinetics. This diversity of bioluminescence colors and patterns suggests that light production in annelids might be involved in a variety of different functions, including defensive mechanisms like sacrificial lures or aposematic signals, and intraspecific communication systems. In this review, we explore the world of luminous annelids, particularly focusing on the current knowledge regarding their taxonomic and ecological diversity and discussing the putative functions and chemistries of their bioluminescent systems. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology 2017. This work is written by US Government employees and is in the public domain in the US.

Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

NASA Astrophysics Data System (ADS)

Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

2004-06-01

Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

Far red bioluminescence from two deep-sea fishes.

PubMed

Widder, E A; Latz, M I; Herring, P J; Case, J F

1984-08-03

Spectral measurements of red bioluminescence were obtained from the deep-sea stomiatoid fishes Aristostomias scintillans (Gilbert) and Malacosteus niger (Ayres). Red luminescence from suborbital light organs extends to the near infrared, with peak emission at approximately 705 nanometers in the far red. These fishes also have postorbital light organs that emit blue luminescence with maxima between 470 and 480 nanometers. The red bioluminescence may be due to an energy transfer system and wavelength-selective filtering.

Chemiluminescence and bioluminescence microbe detection

NASA Technical Reports Server (NTRS)

Taylor, R. E.; Chappelle, E.; Picciolo, G. L.; Jeffers, E. L.; Thomas, R. R.

1978-01-01

Automated biosensors for online use with NASA Water Monitoring System employs bioluminescence and chemiluminescence techniques to rapidly measure microbe contamination of water samples. System eliminates standard laboratory procedures requiring time duration of 24 hours or longer.

Novel bioluminescent coelenterazine derivatives with imidazopyrazinone C-6 extended substitution for Renilla luciferase.

PubMed

Jiang, Tianyu; Yang, Xiaofeng; Yang, Xingye; Yuan, Mingliang; Zhang, Tianchao; Zhang, Huateng; Li, Minyong

2016-06-21

Two series of novel coelenterazine analogues (alkynes and triazoles) with imidazopyrazinone C-6 extended substitution have been designed and synthesized successfully for the extension of bioluminescent substrates. After extensive evaluation, some compounds display excellent bioluminescence properties compared with DeepBlueC in cellulo, thus becoming potential molecules for bioluminescence techniques.

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs.

PubMed

Lu, Yongbo; Kamel-El Sayed, Suzan A; Wang, Kun; Tiede-Lewis, LeAnn M; Grillo, Michael A; Veno, Patricia A; Dusevich, Vladimir; Phillips, Charlotte L; Bonewald, Lynda F; Dallas, Sarah L

2018-06-01

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Shan; Wang, Ming-Wei; Yao, Xiaoqiang

2009-05-15

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newlymore » developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.« less

Midwater Bioluminescence Assessment in the West Alboran Gyre (Mediterranean Sea)

DTIC Science & Technology

1991-09-01

of bioluminescence.I KEYWORDS I ALBORAN SEA JOHNSON-SEA-LINK BATHYPHOTOMETER MEDUSAE BIOLUMINESCENCE SEWARD JOHNSON I RV CTENOPHORES SIPHONOPHORES ...30 C tenophores................................................................ 32 Siphonophores ...good, however, for ctenophores, salps, siphonophores and medusae. Table 2 Specimen No. Genus, Spec!es Taxon I 1896 12 Sappharina sp. Copepod 1896 32b

Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques.

PubMed

Xia, Jixiang; Martinez, Angela; Daniell, Henry; Ebert, Steven N

2011-06-02

Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods. Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that different tissues show different

REVIEW ARTICLE: Bioluminescent signals and the role of reflectors

NASA Astrophysics Data System (ADS)

Herring, Peter J.

2000-11-01

Organisms in a well lit environment use optical signals derived from the selective reflection of ambient light. In a dim or dark environment it is very difficult (because of low photon numbers) to detect the contrast between light reflected from the organism and that from the background, and many organisms use bioluminescent signals instead. The use of such signals on land is largely restricted to sexual signalling by the luminous beetles, but in the deep ocean their use is widespread, involving both many different organisms and a range of uses which parallel those of reflective signals on land. Some bioluminescent signals rely almost entirely on an optically unmodified light source (e.g. a secretion) but others depend upon complex optical structures, particularly reflectors, in the light-emitting organs. Reflectors in the light organs of many shrimp, squid and fish are based on constructive interference systems but employ different biological materials. They and other structures modify the angular, spectral and intensity distributions of bioluminescent signals. The ready availability of highly efficient biological reflectors has been a formative influence in the evolution of bioluminescent signalling in the sea.

A High Sensitivity Bio Photosensor for Detecting a Luciferase Bioluminescence

NASA Astrophysics Data System (ADS)

Kameda, Seiji; Moriyama, Yusuke; Noda, Kenichi; Iwata, Atsushi

A high sensitivity CMOS bio photosensor applicable to a bioluminescent assay was developed with a 0.18µm CMOS image sensor (CIS) process. The bio photosensor consisting of a photosensor and a PWM 20bit A/D converter achieved high sensitivity for detecting a extremely low bioluminescence due to a large photodiode area, a long exposure time and the other noise reduction techniques. The bio photosensor chip has a 2×4 sensor array on a 2.45×2.45mm2 die. Experimental results with the bioluminescence showed the chip can detect below 10-5lux luminescence at room temperature and the power consumption is 32µW.

Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait

PubMed Central

Martini, Séverine; Haddock, Steven H. D.

2017-01-01

The capability of animals to emit light, called bioluminescence, is considered to be a major factor in ecological interactions. Because it occurs across diverse taxa, measurements of bioluminescence can be powerful to detect and quantify organisms in the ocean. In this study, 17 years of video observations were recorded by remotely operated vehicles during surveys off the California Coast, from the surface down to 3,900 m depth. More than 350,000 observations are classified for their bioluminescence capability based on literature descriptions. The organisms represented 553 phylogenetic concepts (species, genera or families, at the most precise taxonomic level defined from the images), distributed within 13 broader taxonomic categories. The importance of bioluminescent marine taxa is highlighted in the water column, as we showed that 76% of the observed individuals have bioluminescence capability. More than 97% of Cnidarians were bioluminescent, and 9 of the 13 taxonomic categories were found to be bioluminescent dominant. The percentage of bioluminescent animals is remarkably uniform over depth. Moreover, the proportion of bioluminescent and non-bioluminescent animals within taxonomic groups changes with depth for Ctenophora, Scyphozoa, Chaetognatha, and Crustacea. Given these results, bioluminescence has to be considered an important ecological trait from the surface to the deep-sea. PMID:28374789

Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait

NASA Astrophysics Data System (ADS)

Martini, Séverine; Haddock, Steven H. D.

2017-04-01

The capability of animals to emit light, called bioluminescence, is considered to be a major factor in ecological interactions. Because it occurs across diverse taxa, measurements of bioluminescence can be powerful to detect and quantify organisms in the ocean. In this study, 17 years of video observations were recorded by remotely operated vehicles during surveys off the California Coast, from the surface down to 3,900 m depth. More than 350,000 observations are classified for their bioluminescence capability based on literature descriptions. The organisms represented 553 phylogenetic concepts (species, genera or families, at the most precise taxonomic level defined from the images), distributed within 13 broader taxonomic categories. The importance of bioluminescent marine taxa is highlighted in the water column, as we showed that 76% of the observed individuals have bioluminescence capability. More than 97% of Cnidarians were bioluminescent, and 9 of the 13 taxonomic categories were found to be bioluminescent dominant. The percentage of bioluminescent animals is remarkably uniform over depth. Moreover, the proportion of bioluminescent and non-bioluminescent animals within taxonomic groups changes with depth for Ctenophora, Scyphozoa, Chaetognatha, and Crustacea. Given these results, bioluminescence has to be considered an important ecological trait from the surface to the deep-sea.

Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

PubMed Central

2011-01-01

Background Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors. Results The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth. Conclusion Lentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to be expressed from multi

Tracking Bioluminescent ETEC during In vivo BALB/c Mouse Colonization

PubMed Central

Rodea, Gerardo E.; Montiel-Infante, Francisco X.; Cruz-Córdova, Ariadnna; Saldaña-Ahuactzi, Zeus; Ochoa, Sara A.; Espinosa-Mazariego, Karina; Hernández-Castro, Rigoberto; Xicohtencatl-Cortes, Juan

2017-01-01

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea worldwide. Adhesion to the human intestinal tract is crucial for colonization. ETEC adhesive structures have been extensively studied; however, colonization dynamics remain uncharacterized. The aim of this study was to track bioluminescent ETEC during in vivo infection. The promoter region of dnaK was fused with the luc gene, resulting in the pRMkluc vector. E. coli K-12 and ETEC FMU073332 strains were electroporated with pRMkluc. E. coli K-12 pRMkluc was bioluminescent; in contrast, the E. coli K-12 control strain did not emit bioluminescence. The highest light emission was measured at 1.9 OD600 (9 h) and quantified over time. The signal was detected starting at time 0 and up to 12 h. Streptomycin-treated BALB/c mice were orogastrically inoculated with either ETEC FMU073332 pRMkluc or E. coli K-12 pRMkluc (control), and bacterial colonization was determined by measuring bacterial shedding in the feces. ETEC FMU073332 pRMkluc shedding started and stopped after inoculation of the control strain, indicating that mouse intestinal colonization by ETEC FMU073332 pRMkluc lasted longer than colonization by the control. The bioluminescence signal of ETEC FMU073332 pRMkluc was captured starting at the time of inoculation until 12 h after inoculation. The bioluminescent signal emitted by ETEC FMU073332 pRMkluc in the proximal mouse ileum was located, and the control signal was identified in the cecum. The detection of maximal light emission and bioluminescence duration allowed us to follow ETEC during in vivo infection. ETEC showed an enhanced colonization and tropism in the mouse intestine compared with those in the control strain. Here, we report the first study of ETEC colonization in the mouse intestine accompanied by in vivo imaging. PMID:28560186

A panel of Trypanosoma brucei strains tagged with blue and red-shifted luciferases for bioluminescent imaging in murine infection models.

PubMed

Van Reet, Nick; Van de Vyver, Hélène; Pyana, Patient Pati; Van der Linden, Anne Marie; Büscher, Philippe

2014-08-01

Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients. We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT. We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better kinetics of their substrate D-luciferin.

A Panel of Trypanosoma brucei Strains Tagged with Blue and Red-Shifted Luciferases for Bioluminescent Imaging in Murine Infection Models

PubMed Central

Van Reet, Nick; Van de Vyver, Hélène; Pyana, Patient Pati; Van der Linden, Anne Marie; Büscher, Philippe

2014-01-01

Background Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients. Methodology/Principal findings We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT. Conclusions/Significance We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better

Transfection microarray and the applications.

PubMed

Miyake, Masato; Yoshikawa, Tomohiro; Fujita, Satoshi; Miyake, Jun

2009-05-01

Microarray transfection has been extensively studied for high-throughput functional analysis of mammalian cells. However, control of efficiency and reproducibility are the critical issues for practical use. By using solid-phase transfection accelerators and nano-scaffold, we provide a highly efficient and reproducible microarray-transfection device, "transfection microarray". The device would be applied to the limited number of available primary cells and stem cells not only for large-scale functional analysis but also reporter-based time-lapse cellular event analysis.

Analytical Applications of Bioluminescence and Chemiluminescence

NASA Technical Reports Server (NTRS)

Chappelle, E. W. (Editor); Picciolo, G. L. (Editor)

1975-01-01

Bioluminescence and chemiluminescence studies were used to measure the amount of adenosine triphosphate and therefore the amount of energy available. Firefly luciferase - luciferin enzyme system was emphasized. Photometer designs are also considered.

Effects of salinity, pH and temperature on the re-establishment of bioluminescence and copper or SDS toxicity in the marine dinoflagellate Pyrocystis lunula using bioluminescence as an endpoint

USGS Publications Warehouse

Craig, J.M.; Klerks, P.L.; Heimann, K.; Waits, J.L.

2003-01-01

Pyrocystis lunula is a unicellular, marine, photoautotrophic, bioluminescent dinoflagellate. This organism is used in the Lumitox ?? bioassay with inhibition of bioluminescence re-establishment as the endpoint. Experiments determined if acute changes in pH, salinity, or temperature had an effect on the organisms' ability to re-establish bioluminescence, or on the bioassay's potential to detect sodium dodecyl sulfate (SDS) and copper toxicity. The re-establishment of bioluminescence itself was not very sensitive to changes in pH within the pH 6-10 range, though reducing pH from 8 to levels below 6 decreased this capacity. Increasing the pH had little effect on Cu or SDS toxicity, but decreasing the pH below 7 virtually eliminated the toxicity of either compound in the bioassay. Lowering the salinity from 33 to 27??? or less resulted in a substantial decrease in re-establishment of bioluminescence, while increasing the salinity to 43 or 48 ??? resulted in a small decline. Salinity had little influence on the bioassay's quantification of Cu toxicity, while the data showed a weak negative relationship between SDS toxicity and salinity. Re-establishment of bioluminescence showed a direct dependence on temperature, but only at 10??C did temperature have an obvious effect on the toxicity of Cu in this bioassay. ?? 2003 Elsevier Science Ltd. All rights reserved.

POLYPHYLY OF NON-BIOLUMINESCENT VIBRIO FISCHERI SHARING A LUX-LOCUS DELETION

PubMed Central

Wollenberg, M.S.; Preheim, S.P.; Polz, M.F.; Ruby, E. G.

2013-01-01

SUMMARY This study reports the first description and molecular characterization of naturally occurring, non-bioluminescent strains of V. fischeri. These ‘dark’ V. fischeri strains remained non-bioluminescent even after treatment with both autoinducer and aldehyde, substrate additions that typically maximize light-production in dim strains of luminous bacteria. Surprisingly, the entire lux locus (8 genes) was absent in over 97% of these dark V. fischeri strains. Although these strains were all collected from a Massachusetts (USA) estuary in 2007, phylogenetic reconstructions allowed us to reject the hypothesis that these newly described non-bioluminescent strains exhibit monophyly within the V. fischeri clade. These dark strains exhibited a competitive disadvantage against native bioluminescent strains when colonizing the light organ of the model V. fischeri host, the Hawaiian bobtail squid Euprymna scolopes. Significantly, we believe that the data collected in this study may suggest the first observation of a functional, parallel locus-deletion event among independent lineages of a non-pathogenic bacterial species. PMID:21980988

Bioluminescent bioreporter integrated circuit

DOEpatents

Simpson, Michael L.; Sayler, Gary S.; Paulus, Michael J.

2000-01-01

Disclosed are monolithic bioelectronic devices comprising a bioreporter and an OASIC. These bioluminescent bioreporter integrated circuit are useful in detecting substances such as pollutants, explosives, and heavy-metals residing in inhospitable areas such as groundwater, industrial process vessels, and battlefields. Also disclosed are methods and apparatus for environmental pollutant detection, oil exploration, drug discovery, industrial process control, and hazardous chemical monitoring.

Formation of temperature front in stably stratified turbulence

NASA Astrophysics Data System (ADS)

Kimura, Yoshifumi; Sullivan, Peter; Herring, Jackson

2016-11-01

An important feature of stably stratified turbulence is the significant influence of internal gravity waves which makes stably stratified turbulence unique compared to homogeneous isotropic turbulence. In this paper, we investigate the genesis of temperature fronts-a crucial subject both practically and fundamentally-in stably stratified turbulence using Direct Numerical Simulations (DNS) of the Navier-Stokes equation under the Boussinesq approximation with 10243 grid points. Vertical profiles of temperature fluctuations show almost vertically periodic sawtooth wavy structures with negative and positive layers stacked together with clear boundaries implying a sharp temperature fronts. The sawtooth waves consist of gradual decreasing temperature fluctuations with rapid recovery to a positive value as the frontal boundary is crossed vertically. This asymmetry of gradients comes from the structure that warm temperature region lies on top of cool temperature region, and can be verified in the skewed probability density function (PDF) of vertical temperature gradient. We try to extract the flow structures and mechanism for the formation and maintenance of the strong temperature front numerically.

Bioluminescent system for dynamic imaging of cell and animal behavior

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hara-Miyauchi, Chikako; Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, Saitama 351-0198; Department of Biophysics and Biochemistry, Graduate School of Health Care Sciences, Tokyo Medical and Dental University, Tokyo 113-8510

2012-03-09

Highlights: Black-Right-Pointing-Pointer We combined a yellow variant of GFP and firefly luciferase to make ffLuc-cp156. Black-Right-Pointing-Pointer ffLuc-cp156 showed improved photon yield in cultured cells and transgenic mice. Black-Right-Pointing-Pointer ffLuc-cp156 enabled video-rate bioluminescence imaging of freely-moving animals. Black-Right-Pointing-Pointer ffLuc-cp156 mice enabled tracking real-time drug delivery in conscious animals. -- Abstract: The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over threemore » orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies.« less

An autonomous vehicle approach for quantifying bioluminescence in ports and harbors

NASA Astrophysics Data System (ADS)

Moline, Mark; Bissett, Paul; Blackwell, Shelley; Mueller, James; Sevadjian, Jeff; Trees, Charles; Zaneveld, Ron

2005-05-01

Bioluminescence emitted from marine organisms upon mechanical stimulation is an obvious military interest, as it provides a low-tech method of identifying surface and subsurface vehicles and swimmer tracks. Clearly, the development of a passive method of identifying hostile ships, submarines, and swimmers, as well as the development of strategies to reduce the risk of detection by hostile forces is relevant to Naval operations and homeland security. The measurement of bioluminescence in coastal waters has only recently received attention as the platforms and sensors were not scaled for the inherent small-scale nature of nearshore environments. In addition to marine forcing, many ports and harbors are influenced by freshwater inputs, differential density layering and higher turbidity. The spatial and temporal fluctuations of these optical water types overlaid on changes in the bioluminescence potential make these areas uniquely complex. The development of an autonomous underwater vehicle with a bioluminescence capability allows measurements on sub-centimeter horizontal and vertical scales in shallow waters and provides the means to map the potential for detection of moving surface or subsurface objects. A deployment in San Diego Bay shows the influence of tides on the distribution of optical water types and the distribution of bioluminescent organisms. Here, these data are combined to comment on the potential for threat reduction in ports and harbors.

In vivo bioluminescence and reflectance imaging of multiple organs in bioluminescence reporter mice by bundled-fiber-coupled microscopy

PubMed Central

Ando, Yoriko; Sakurai, Takashi; Koida, Kowa; Tei, Hajime; Hida, Akiko; Nakao, Kazuki; Natsume, Mistuo; Numano, Rika

2016-01-01

Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner. PMID:27231601

Hv 1 Proton Channels in Dinoflagellates: Not Just for Bioluminescence?

PubMed

Kigundu, Gabriel; Cooper, Jennifer L; Smith, Susan M E

2018-04-26

Bioluminescence in dinoflagellates is controlled by H V 1 proton channels. Database searches of dinoflagellate transcriptomes and genomes yielded hits with sequence features diagnostic of all confirmed H V 1, and show that H V 1 is widely distributed in the dinoflagellate phylogeny including the basal species Oxyrrhis marina. Multiple sequence alignments followed by phylogenetic analysis revealed three major subfamilies of H V 1 that do not correlate with presence of theca, autotrophy, geographic location, or bioluminescence. These data suggest that most dinoflagellates express a H V 1 which has a function separate from bioluminescence. Sequence evidence also suggests that dinoflagellates can contain more than one H V 1 gene. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Biological water quality monitoring using chemiluminescent and bioluminescent techniques

NASA Technical Reports Server (NTRS)

Thomas, R. R.

1978-01-01

Automated chemiluminescence and bioluminescence sensors were developed for the continuous monitoring of microbial levels in water supplies. The optimal chemical procedures were determined for the chemiluminescence system to achieve maximum sensitivity. By using hydrogen peroxide, reaction rate differentiation, ethylene diamine tetraacetic acid (EDTA), and carbon monoxide pretreatments, factors which cause interference were eliminated and specificity of the reaction for living and dead bacteria was greatly increased. By employing existing technology with some modifications, a sensitive and specific bioluminescent system was developed.

Markedly Enhanced Skeletal Muscle Transfection Achieved by the Ultrasound-Targeted Delivery of Non-Viral Gene Nanocarriers with Microbubbles

PubMed Central

Burke, Caitlin W.; Suk, Jung Soo; Kim, Anthony J.; Hsiang, Yu-Han J.; Klibanov, Alexander L.; Hanes, Justin; Price, Richard J.

2012-01-01

Our goal was to enhance ultrasound (US)-targeted skeletal muscle transfection through the use of poly(ethyleneglycol) (PEG)/polyethylenimine (PEI) nanocomplex gene carriers and adjustments to US and microbubble (MB) parameters. C57BL/6 mice received an intravenous infusion of MBs and either “naked” luciferase plasmid or luciferase plasmid condensed in PEG/PEI nanocomplexes. Pulsed ultrasound (1MHz; 0.6 MPa or 0.8 MPa) was applied to the right hindlimb for 12 mins. Luciferase activity in both hindlimbs was assessed at 3, 5, 7, and 10 days post-treatment by bioluminescent imaging. When targeted to hindlimb using unsorted MBs and 0.6 MPa US, 7 days after treatment, we observed a >60-fold increase in luciferase activity in PEG/PEI nanocomplex treated muscles over muscles treated with “naked” plasmid DNA. Luciferase activity was consistently greater after treatment with PEG/PEI nanocomplexes at 0.6 MPa as compared to 0.8 MPa. The combination of small diameter MBs and 0.6 MPa US also resulted in significantly greater gene expression when compared to concentration matched intramuscular injections, a control condition in which considerably more PEG/PEI nanocomplexes were present in tissue. This result suggests that, in addition to facilitating PEG/PEI nanocomplex delivery from the bloodstream to tissue, US enhances transfection via one or more secondary mechanisms, including increased cellular uptake and/or trafficking to the nucleus of PEG/PEI nanocomplexes. We conclude that PEG/PEI nanocomplexes may be used to markedly enhance the amplitude of US-MB-targeted skeletal muscle transfection and that activating “small” MBs with a moderate level (0.6 MPa) of acoustic pressure can further enhance these effects. PMID:22800583

Modeling bioluminescent photon transport in tissue based on Radiosity-diffusion model

NASA Astrophysics Data System (ADS)

Sun, Li; Wang, Pu; Tian, Jie; Zhang, Bo; Han, Dong; Yang, Xin

2010-03-01

Bioluminescence tomography (BLT) is one of the most important non-invasive optical molecular imaging modalities. The model for the bioluminescent photon propagation plays a significant role in the bioluminescence tomography study. Due to the high computational efficiency, diffusion approximation (DA) is generally applied in the bioluminescence tomography. But the diffusion equation is valid only in highly scattering and weakly absorbing regions and fails in non-scattering or low-scattering tissues, such as a cyst in the breast, the cerebrospinal fluid (CSF) layer of the brain and synovial fluid layer in the joints. A hybrid Radiosity-diffusion model is proposed for dealing with the non-scattering regions within diffusing domains in this paper. This hybrid method incorporates a priori information of the geometry of non-scattering regions, which can be acquired by magnetic resonance imaging (MRI) or x-ray computed tomography (CT). Then the model is implemented using a finite element method (FEM) to ensure the high computational efficiency. Finally, we demonstrate that the method is comparable with Mont Carlo (MC) method which is regarded as a 'gold standard' for photon transportation simulation.

Polyphyly of non-bioluminescent Vibrio fischeri sharing a lux-locus deletion.

PubMed

Wollenberg, M S; Preheim, S P; Polz, M F; Ruby, E G

2012-03-01

This study reports the first description and molecular characterization of naturally occurring, non-bioluminescent strains of Vibrio fischeri. These 'dark' V. fischeri strains remained non-bioluminescent even after treatment with both autoinducer and aldehyde, substrate additions that typically maximize light production in dim strains of luminous bacteria. Surprisingly, the entire lux locus (eight genes) was absent in over 97% of these dark V. fischeri strains. Although these strains were all collected from a Massachusetts (USA) estuary in 2007, phylogenetic reconstructions allowed us to reject the hypothesis that these newly described non-bioluminescent strains exhibit monophyly within the V. fischeri clade. These dark strains exhibited a competitive disadvantage against native bioluminescent strains when colonizing the light organ of the model V. fischeri host, the Hawaiian bobtail squid Euprymna scolopes. Significantly, we believe that the data collected in this study may suggest the first observation of a functional, parallel locus-deletion event among independent lineages of a non-pathogenic bacterial species. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

Three-dimensional multi bioluminescent sources reconstruction based on adaptive finite element method

NASA Astrophysics Data System (ADS)

Ma, Xibo; Tian, Jie; Zhang, Bo; Zhang, Xing; Xue, Zhenwen; Dong, Di; Han, Dong

2011-03-01

Among many optical molecular imaging modalities, bioluminescence imaging (BLI) has more and more wide application in tumor detection and evaluation of pharmacodynamics, toxicity, pharmacokinetics because of its noninvasive molecular and cellular level detection ability, high sensitivity and low cost in comparison with other imaging technologies. However, BLI can not present the accurate location and intensity of the inner bioluminescence sources such as in the bone, liver or lung etc. Bioluminescent tomography (BLT) shows its advantage in determining the bioluminescence source distribution inside a small animal or phantom. Considering the deficiency of two-dimensional imaging modality, we developed three-dimensional tomography to reconstruct the information of the bioluminescence source distribution in transgenic mOC-Luc mice bone with the boundary measured data. In this paper, to study the osteocalcin (OC) accumulation in transgenic mOC-Luc mice bone, a BLT reconstruction method based on multilevel adaptive finite element (FEM) algorithm was used for localizing and quantifying multi bioluminescence sources. Optical and anatomical information of the tissues are incorporated as a priori knowledge in this method, which can reduce the ill-posedness of BLT. The data was acquired by the dual modality BLT and Micro CT prototype system that was developed by us. Through temperature control and absolute intensity calibration, a relative accurate intensity can be calculated. The location of the OC accumulation was reconstructed, which was coherent with the principle of bone differentiation. This result also was testified by ex vivo experiment in the black 96-plate well using the BLI system and the chemiluminescence apparatus.

A two-hour antibiotic susceptibility test by ATP-bioluminescence.

PubMed

March Rosselló, Gabriel Alberto; García-Loygorri Jordán de Urries, María Cristina; Gutiérrez Rodríguez, María Purificación; Simarro Grande, María; Orduña Domingo, Antonio; Bratos Pérez, Miguel Ángel

2016-01-01

The antibiotic susceptibility test (AST) in Clinical Microbiology laboratories is still time-consuming, and most procedures take 24h to yield results. In this study, a rapid antimicrobial susceptibility test using ATP-bioluminescence has been developed. The design of method was performed using five ATCC collection strains of known susceptibility. This procedure was then validated against standard commercial methods on 10 strains of enterococci, 10 staphylococci, 10 non-fermenting gram negative bacilli, and 13 Enterobacteriaceae from patients. The agreement obtained in the sensitivity between the ATP-bioluminescence method and commercial methods (E-test, MicroScan and VITEK2) was 100%. In summary, the preliminary results obtained in this work show that the ATP-bioluminescence method could provide a fast and reliable AST in two hours. Copyright © 2015 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Comparison of the thermostability of recombinant luciferases from Brazilian bioluminescent beetles: Relationship with kinetics and bioluminescence colours.

PubMed

Oliveira, Gabriela; Viviani, Vadim R

2018-03-01

Firefly luciferases have been used extensively as bioanalytical reagents and their cDNAs as reporter genes for biosensors and bioimaging, but they are in general unstable at temperatures above 30°C. In the past few years, efforts have been made to stabilize some firefly luciferases for better application as analytical reagents. Novel luciferases from different beetle families, displaying distinct bioluminescence colours and kinetics, may offer desirable alternatives to extend the range of applications. In the past years, our group has cloned the largest variety of luciferases from the three main families of bioluminescent beetles (Elateridae: P. termitilluminans, F. bruchi, P. angustus; Phengodidae: P. hirtus, P. vivianii; and Lampyridae: A. vivianii, C. distinctus and Macrolampis sp2) occurring in Brazilian biomes. We compared the thermostability of these recombinant luciferases and investigated their relationships with bioluminescence spectra and kinetics. The most thermostable luciferases were those of Pyrearinus termitilluminans larval click beetle (534 nm), Amydetes vivianii firefly (539 nm) and Phrixotrix vivianii railroad worm (546 nm), which are the most blue-shifted examples in each family, confirming the trend that the most blue-shifted emitting luciferases are also the most thermostable. Comparatively, commercial P. pyralis firefly luciferase was less thermostable than P. termitilluminans click beetle and A. vivianii firefly luciferases. The higher thermostability in these luciferases could be related to higher degree of hydrophobic packing and disulfide bond content (for firefly luciferases). Copyright © 2017 John Wiley & Sons, Ltd.

A synthetic luciferin improves in vivo bioluminescence imaging of gene expression in cardiovascular brain regions.

PubMed

Simonyan, Hayk; Hurr, Chansol; Young, Colin N

2016-10-01

Bioluminescence imaging is an effective tool for in vivo investigation of molecular processes. We have demonstrated the applicability of bioluminescence imaging to spatiotemporally monitor gene expression in cardioregulatory brain nuclei during the development of cardiovascular disease, via incorporation of firefly luciferase into living animals, combined with exogenous d-luciferin substrate administration. Nevertheless, d-luciferin uptake into the brain tissue is low, which decreases the sensitivity of bioluminescence detection, particularly when considering small changes in gene expression in tiny central areas. Here, we tested the hypothesis that a synthetic luciferin, cyclic alkylaminoluciferin (CycLuc1), would be superior to d-luciferin for in vivo bioluminescence imaging in cardiovascular brain regions. Male C57B1/6 mice underwent targeted delivery of an adenovirus encoding the luciferase gene downstream of the CMV promoter to the subfornical organ (SFO) or paraventricular nucleus of hypothalamus (PVN), two crucial cardioregulatory neural regions. While bioluminescent signals could be obtained following d-luciferin injection (150 mg/kg), CycLuc1 administration resulted in a three- to fourfold greater bioluminescent emission from the SFO and PVN, at 10- to 20-fold lower substrate concentrations (7.5-15 mg/kg). This CycLuc1-mediated enhancement in bioluminescent emission was evident early following substrate administration (i.e., 6-10 min) and persisted for up to 1 h. When the exposure time was reduced from 60 s to 1,500 ms, minimal signal in the PVN was detectable with d-luciferin, whereas bioluminescent images could be reliably captured with CycLuc1. These findings demonstrate that bioluminescent imaging with the synthetic luciferin CycLuc1 provides an improved physiological genomics tool to investigate molecular events in discrete cardioregulatory brain nuclei. Copyright © 2016 the American Physiological Society.

Molecular detection of bioluminescent dinoflagellates in surface waters of the Patagonian shelf during early austral summer 2008.

PubMed

Valiadi, Martha; Painter, Stuart C; Allen, John T; Balch, William M; Iglesias-Rodriguez, M Debora

2014-01-01

We investigated the distribution of bioluminescent dinoflagellates in the Patagonian Shelf region using "universal" PCR primers for the dinoflagellate luciferase gene. Luciferase gene sequences and single cell PCR tests, in conjunction with taxonomic identification by microscopy, allowed us to identify and quantify bioluminescent dinoflagellates. We compared these data to coincidental discrete optical measurements of stimulable bioluminescence intensity. Molecular detection of the luciferase gene showed that bioluminescent dinoflagellates were widespread across the majority of the Patagonian Shelf region. Their presence was comparatively underestimated by optical bioluminescence measurements, whose magnitude was affected by interspecific differences in bioluminescence intensity and by the presence of other bioluminescent organisms. Molecular and microscopy data showed that the complex hydrography of the area played an important role in determining the distribution and composition of dinoflagellate populations. Dinoflagellates were absent south of the Falkland Islands where the cold, nutrient-rich, and well-mixed waters of the Falklands Current favoured diatoms instead. Diverse populations of dinoflagellates were present in the warmer, more stratified waters of the Patagonian Shelf and Falklands Current as it warmed northwards. Here, the dinoflagellate population composition could be related to distinct water masses. Our results provide new insight into the prevalence of bioluminescent dinoflagellates in Patagonian Shelf waters and demonstrate that a molecular approach to the detection of bioluminescent dinoflagellates in natural waters is a promising tool for ecological studies of these organisms.

Molecular Detection of Bioluminescent Dinoflagellates in Surface Waters of the Patagonian Shelf during Early Austral Summer 2008

PubMed Central

Valiadi, Martha; Painter, Stuart C.; Allen, John T.; Balch, William M.; Iglesias-Rodriguez, M. Debora

2014-01-01

We investigated the distribution of bioluminescent dinoflagellates in the Patagonian Shelf region using “universal” PCR primers for the dinoflagellate luciferase gene. Luciferase gene sequences and single cell PCR tests, in conjunction with taxonomic identification by microscopy, allowed us to identify and quantify bioluminescent dinoflagellates. We compared these data to coincidental discrete optical measurements of stimulable bioluminescence intensity. Molecular detection of the luciferase gene showed that bioluminescent dinoflagellates were widespread across the majority of the Patagonian Shelf region. Their presence was comparatively underestimated by optical bioluminescence measurements, whose magnitude was affected by interspecific differences in bioluminescence intensity and by the presence of other bioluminescent organisms. Molecular and microscopy data showed that the complex hydrography of the area played an important role in determining the distribution and composition of dinoflagellate populations. Dinoflagellates were absent south of the Falkland Islands where the cold, nutrient-rich, and well-mixed waters of the Falklands Current favoured diatoms instead. Diverse populations of dinoflagellates were present in the warmer, more stratified waters of the Patagonian Shelf and Falklands Current as it warmed northwards. Here, the dinoflagellate population composition could be related to distinct water masses. Our results provide new insight into the prevalence of bioluminescent dinoflagellates in Patagonian Shelf waters and demonstrate that a molecular approach to the detection of bioluminescent dinoflagellates in natural waters is a promising tool for ecological studies of these organisms. PMID:24918444

Reverse Transfection Using Gold Nanoparticles

NASA Astrophysics Data System (ADS)

Yamada, Shigeru; Fujita, Satoshi; Uchimura, Eiichiro; Miyake, Masato; Miyake, Jun

Reverse transfection from a solid surface has the potential to deliver genes into various types of cell and tissue more effectively than conventional methods of transfection. We present a method for reverse transfection using a gold colloid (GC) as a nanoscaffold by generating nanoclusters of the DNA/reagentcomplex on a glass surface, which could then be used for the regulation of the particle size of the complex and delivery of DNA into nuclei. With this method, we have found that the conjugation of gold nanoparticles (20 nm in particle size) to the pEGFP-N1/Jet-PEI complex resulted in an increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of transfection) from human mesenchymal stem cells (hMSCs), as compared with the control without GC. In this manner, we constructed a method for reverse transfection using GC to deliver genes into the cells effectively.

Bioluminescent Antibodies for Point‐of‐Care Diagnostics

PubMed Central

Xue, Lin; Yu, Qiuliyang; Griss, Rudolf; Schena, Alberto

2017-01-01

Abstract We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no‐wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP‐tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR max>500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera. PMID:28510347

Cloning and characterization of new bioluminescent proteins

NASA Astrophysics Data System (ADS)

Szent-Gyorgyi, Christopher; Ballou, Byron T.; Dagnal, Erich; Bryan, Bruce

1999-07-01

Over the past two years Prolume has undertaken a comprehensive program to clone luciferases and associated 'green fluorescent proteins' (GFPs) from marine animals that use coelenterazine as the luciferin. To data we have cloned several bioluminescent proteins, including two novel copepod luciferases and two anthozoan GFPs. These four proteins have sequences that differ greatly form previously cloned analogous proteins; the sequence diversity apparently is due to independent evolutionary origins and unusual evolutionary constraints. Thus coelenterazine-based bioluminescent systems may also manifest a variety of useful properties. We discuss form this taxonomic perspective the initial biochemical and spectral characterization of our cloned proteins. Emphasis is placed on the anthozoan luciferase-GFP systems, whose efficient resonance energy transfer has elicited much current interest.

Bacterial bioluminescence as a lure for marine zooplankton and fish.

PubMed

Zarubin, Margarita; Belkin, Shimshon; Ionescu, Michael; Genin, Amatzia

2012-01-17

The benefits of bioluminescence for nonsymbiotic marine bacteria have not been elucidated fully. One of the most commonly cited explanations, proposed more than 30 y ago, is that bioluminescence augments the propagation and dispersal of bacteria by attracting fish to consume the luminous material. This hypothesis, based mostly on the prevalence of luminous bacteria in fish guts, has not been tested experimentally. Here we show that zooplankton that contacts and feeds on the luminescent bacterium Photobacterium leiognathi starts to glow, and demonstrate by video recordings that glowing individuals are highly vulnerable to predation by nocturnal fish. Glowing bacteria thereby are transferred to the nutritious guts of fish and zooplankton, where they survive digestion and gain effective means for growth and dispersal. Using bioluminescence as bait appears to be highly beneficial for marine bacteria, especially in food-deprived environments of the deep sea.

Evidence for light perception in a bioluminescent organ

PubMed Central

Tong, Deyan; Rozas, Natalia S.; Oakley, Todd H.; Mitchell, Jane; Colley, Nansi J.; McFall-Ngai, Margaret J.

2009-01-01

Here we show that bioluminescent organs of the squid Euprymna scolopes possess the molecular, biochemical, and physiological capability for light detection. Transcriptome analyses revealed expression of genes encoding key visual transduction proteins in light-organ tissues, including the same isoform of opsin that occurs in the retina. Electroretinograms demonstrated that the organ responds physiologically to light, and immunocytochemistry experiments localized multiple proteins of visual transduction cascades to tissues housing light-producing bacterial symbionts. These data provide evidence that the light-organ tissues harboring the symbionts serve as extraocular photoreceptors, with the potential to perceive directly the bioluminescence produced by their bacterial partners. PMID:19509343

Bioluminescence as the Basis for the Detection of Trichothecenes

DTIC Science & Technology

1986-03-17

screened for their ability to quench bioluminescence were obtained through the courtesy of Dr. Lou Carson, of the Toxicology Division of the Food and...34 Recent Adv. Phytochem . 9, 167 (1974). 13. Lyman, J. and Fleming, R.H., "Composition of Seawater," J. Mar. Res. 3, 134 (1940). 14. Mayer, C.F., "Endemic...DIELDRIN Cl CI Cl~c C 1 2I• HEPTACHLOR EPOXIDE OCTACHLOR EPOXIDE "Fig. 11 - Pesticides screened for ability to quench bioluminescence Ir £ d, PF K.I IR 10 R 125 - I ’S * N 586 9 -q

Bioorthogonal chemistry in bioluminescence imaging.

PubMed

Godinat, Aurélien; Bazhin, Arkadiy A; Goun, Elena A

2018-05-18

Bioorthogonal chemistry has developed significant over the past few decades, to the particular benefit of molecular imaging. Bioluminescence imaging (BLI) along with other imaging modalities have significantly benefitted from this chemistry. Here, we review bioorthogonal reactions that have been used to signific antly broaden the application range of BLI. Copyright © 2018. Published by Elsevier Ltd.

Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

PubMed

Marivin, E; Mourot, B; Loyer, P; Rime, H; Bobe, J; Fostier, A

2015-09-15

Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes. Copyright © 2015 Elsevier Inc. All rights reserved.

Discovery of a glowing millipede in California and the gradual evolution of bioluminescence in Diplopoda.

PubMed

Marek, Paul E; Moore, Wendy

2015-05-19

The rediscovery of the Californian millipede Xystocheir bistipita surprisingly reveals that the species is bioluminescent. Using molecular phylogenetics, we show that X. bistipita is the evolutionary sister group of Motyxia, the only genus of New World bioluminescent millipedes. We demonstrate that bioluminescence originated in the group's most recent common ancestor and evolved by gradual, directional change through diversification. Because bioluminescence in Motyxia has been experimentally demonstrated to be aposematic, forewarning of the animal's cyanide-based toxins, these results are contrary to aposematic theory and empirical evidence that a warning pattern cannot evolve gradually in unpalatable prey. However, gradual evolution of a warning pattern is plausible if faint light emission served another function and was co-opted as an aposematic signal later in the diversification of the genus. Luminescence in Motyxia stem-group taxa may have initially evolved to cope with reactive oxygen stress triggered by a hot, dry environment and was repurposed for aposematism by high-elevation crown-group taxa colonizing new habitats with varying levels of predation. The discovery of bioluminescence in X. bistipita and its pivotal phylogenetic location provides insight into the independent and repeated evolution of bioluminescence across the tree of life.

Effect of antiangiogenic therapy on luciferase activity in a cytomegalovirus- or HSP70-promoter-transfected M21 tumor model

NASA Astrophysics Data System (ADS)

Hundt, Walter; Schink, Christian; Steinbach, Silke; O'Connell-Rodwell, Caitlin E.; Kiessling, Andreas; Librizzi, Damiano; Burbelko, Mykhaylo; Guccione, Samira

2012-06-01

We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650 mm3. Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8+/-103.4 mm2 at the beginning versus 704.8+/-94.4 mm3 at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9%+/-4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5+/-0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays.

PubMed

Rheiner, Steven; Reichel, Derek; Rychahou, Piotr; Izumi, Tadahide; Yang, Hsin-Sheng; Bae, Younsoo

2017-08-07

Poly(ethylene glycol)-conjugated polyethylenimine (PEG-PEI) is a widely studied cationic polymer used to develop non-viral vectors for siRNA therapy of genetic disorders including cancer. Cell lines stably expressing luciferase reporter protein typically evaluate the transfection efficacy of siRNA/PEG-PEI complexes, however recent findings revealed that PEG-PEI can reduce luciferase expression independent of siRNA. This study elucidates a cause of the false positive effect in luciferase assays by using polymer nanoassemblies (PNAs) made from PEG, PEI, poly-(l-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): PEG-PEI (2P), PEG-PEI-PAL (3P), PEG-PLL (2P'), PEG-PLL-PAL (3P'), and PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of luciferase using a colorectal cancer cell line expressing luciferase (HT29/LUC) concluded that 2P and 2P' caused no luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P'<2PD<3P). Although cell viability remained stagnant, 3P triggered cellular stress responses including increased membrane porosity and decreased ATP and cellular protein concentrations. Raman spectroscopy suggested that hydrophobic groups influence PNA conformation changes, which may have caused over-ubiquitination and degradation of luciferase in the cells. These results indicate that hydrophobically modified PEG-PEI induces cellular distress causing over-ubiquitination of the luciferase protein, producing false positive siRNA transfection in the luciferase assay. Copyright © 2017 Elsevier B.V. All rights reserved.

Bacterial bioluminescence as a lure for marine zooplankton and fish

PubMed Central

Zarubin, Margarita; Belkin, Shimshon; Ionescu, Michael; Genin, Amatzia

2012-01-01

The benefits of bioluminescence for nonsymbiotic marine bacteria have not been elucidated fully. One of the most commonly cited explanations, proposed more than 30 y ago, is that bioluminescence augments the propagation and dispersal of bacteria by attracting fish to consume the luminous material. This hypothesis, based mostly on the prevalence of luminous bacteria in fish guts, has not been tested experimentally. Here we show that zooplankton that contacts and feeds on the luminescent bacterium Photobacterium leiognathi starts to glow, and demonstrate by video recordings that glowing individuals are highly vulnerable to predation by nocturnal fish. Glowing bacteria thereby are transferred to the nutritious guts of fish and zooplankton, where they survive digestion and gain effective means for growth and dispersal. Using bioluminescence as bait appears to be highly beneficial for marine bacteria, especially in food-deprived environments of the deep sea. PMID:22203999

Foraging in the Darkness of the Southern Ocean: Influence of Bioluminescence on a Deep Diving Predator

PubMed Central

Vacquié-Garcia, Jade; Royer, François; Dragon, Anne-Cécile; Viviant, Morgane; Bailleul, Frédéric; Guinet, Christophe

2012-01-01

How non-echolocating deep diving marine predators locate their prey while foraging remains mostly unknown. Female southern elephant seals (SES) (Mirounga leonina) have vision adapted to low intensity light with a peak sensitivity at 485 nm. This matches the wavelength of bioluminescence produced by a large range of marine organisms including myctophid fish, SES’s main prey. In this study, we investigated whether bioluminescence provides an accurate estimate of prey occurrence for SES. To do so, four SES were satellite-tracked during their post-breeding foraging trip and were equipped with Time-Depth-Recorders that also recorded light levels every two seconds. A total of 3386 dives were processed through a light-treatment model that detected light events higher than ambient level, i.e. bioluminescence events. The number of bioluminescence events was related to an index of foraging intensity for SES dives deep enough to avoid the influence of natural ambient light. The occurrence of bioluminescence was found to be negatively related to depth both at night and day. Foraging intensity was also positively related to bioluminescence both during day and night. This result suggests that bioluminescence likely provides SES with valuable indications of prey occurrence and might be a key element in predator-prey interactions in deep-dark marine environments. PMID:22952706

Bioluminescent Antibodies for Point-of-Care Diagnostics.

PubMed

Xue, Lin; Yu, Qiuliyang; Griss, Rudolf; Schena, Alberto; Johnsson, Kai

2017-06-12

We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR max >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Numerical modeling of the dynamic response of a bioluminescent bacterial biosensor.

PubMed

Affi, Mahmoud; Solliec, Camille; Legentilhomme, Patrick; Comiti, Jacques; Legrand, Jack; Jouanneau, Sulivan; Thouand, Gérald

2016-12-01

Water quality and water management are worldwide issues. The analysis of pollutants and in particular, heavy metals, is generally conducted by sensitive but expensive physicochemical methods. Other alternative methods of analysis, such as microbial biosensors, have been developed for their potential simplicity and expected moderate cost. Using a biosensor for a long time generates many changes in the growth of the immobilized bacteria and consequently alters the robustness of the detection. This work simulated the operation of a biosensor for the long-term detection of cadmium and improved our understanding of the bioluminescence reaction dynamics of bioreporter bacteria inside an agarose matrix. The choice of the numerical tools is justified by the difficulty to measure experimentally in every condition the biosensor functioning during a long time (several days). The numerical simulation of a biomass profile is made by coupling the diffusion equation and the consumption/reaction of the nutrients by the bacteria. The numerical results show very good agreement with the experimental profiles. The growth model verified that the bacterial growth is conditioned by both the diffusion and the consumption of the nutrients. Thus, there is a high bacterial density in the first millimeter of the immobilization matrix. The growth model has been very useful for the development of the bioluminescence model inside the gel and shows that a concentration of oxygen greater than or equal to 22 % of saturation is required to maintain a significant level of bioluminescence. A continuous feeding of nutrients during the process of detection of cadmium leads to a biofilm which reduces the diffusion of nutrients and restricts the presence of oxygen from the first layer of the agarose (1 mm) and affects the intensity of the bioluminescent reaction. The main advantage of this work is to link experimental works with numerical models of growth and bioluminescence in order to provide a

Photon Hunting in the Twilight Zone: Visual Features of Mesopelagic Bioluminescent Sharks

PubMed Central

Claes, Julien M.; Partridge, Julian C.; Hart, Nathan S.; Garza-Gisholt, Eduardo; Ho, Hsuan-Ching; Mallefet, Jérôme; Collin, Shaun P.

2014-01-01

The mesopelagic zone is a visual scene continuum in which organisms have developed various strategies to optimize photon capture. Here, we used light microscopy, stereology-assisted retinal topographic mapping, spectrophotometry and microspectrophotometry to investigate the visual ecology of deep-sea bioluminescent sharks [four etmopterid species (Etmopterus lucifer, E. splendidus, E. spinax and Trigonognathus kabeyai) and one dalatiid species (Squaliolus aliae)]. We highlighted a novel structure, a translucent area present in the upper eye orbit of Etmopteridae, which might be part of a reference system for counterillumination adjustment or acts as a spectral filter for camouflage breaking, as well as several ocular specialisations such as aphakic gaps and semicircular tapeta previously unknown in elasmobranchs. All species showed pure rod hexagonal mosaics with a high topographic diversity. Retinal specialisations, formed by shallow cell density gradients, may aid in prey detection and reflect lifestyle differences; pelagic species display areae centrales while benthopelagic and benthic species display wide and narrow horizontal streaks, respectively. One species (E. lucifer) displays two areae within its horizontal streak that likely allows detection of conspecifics' elongated bioluminescent flank markings. Ganglion cell topography reveals less variation with all species showing a temporal area for acute frontal binocular vision. This area is dorsally extended in T. kabeyai, allowing this species to adjust the strike of its peculiar jaws in the ventro-frontal visual field. Etmopterus lucifer showed an additional nasal area matching a high rod density area. Peak spectral sensitivities of the rod visual pigments (λmax) fall within the range 484–491 nm, allowing these sharks to detect a high proportion of photons present in their habitat. Comparisons with previously published data reveal ocular differences between bioluminescent and non-bioluminescent deep

Photon hunting in the twilight zone: visual features of mesopelagic bioluminescent sharks.

PubMed

Claes, Julien M; Partridge, Julian C; Hart, Nathan S; Garza-Gisholt, Eduardo; Ho, Hsuan-Ching; Mallefet, Jérôme; Collin, Shaun P

2014-01-01

The mesopelagic zone is a visual scene continuum in which organisms have developed various strategies to optimize photon capture. Here, we used light microscopy, stereology-assisted retinal topographic mapping, spectrophotometry and microspectrophotometry to investigate the visual ecology of deep-sea bioluminescent sharks [four etmopterid species (Etmopterus lucifer, E. splendidus, E. spinax and Trigonognathus kabeyai) and one dalatiid species (Squaliolus aliae)]. We highlighted a novel structure, a translucent area present in the upper eye orbit of Etmopteridae, which might be part of a reference system for counterillumination adjustment or acts as a spectral filter for camouflage breaking, as well as several ocular specialisations such as aphakic gaps and semicircular tapeta previously unknown in elasmobranchs. All species showed pure rod hexagonal mosaics with a high topographic diversity. Retinal specialisations, formed by shallow cell density gradients, may aid in prey detection and reflect lifestyle differences; pelagic species display areae centrales while benthopelagic and benthic species display wide and narrow horizontal streaks, respectively. One species (E. lucifer) displays two areae within its horizontal streak that likely allows detection of conspecifics' elongated bioluminescent flank markings. Ganglion cell topography reveals less variation with all species showing a temporal area for acute frontal binocular vision. This area is dorsally extended in T. kabeyai, allowing this species to adjust the strike of its peculiar jaws in the ventro-frontal visual field. Etmopterus lucifer showed an additional nasal area matching a high rod density area. Peak spectral sensitivities of the rod visual pigments (λmax) fall within the range 484-491 nm, allowing these sharks to detect a high proportion of photons present in their habitat. Comparisons with previously published data reveal ocular differences between bioluminescent and non-bioluminescent deep

COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene

NASA Technical Reports Server (NTRS)

Breault, D. T.; Lichtler, A. C.; Rowe, D. W.

1997-01-01

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

Transformation Experiment Using Bioluminescence Genes of "Vibrio fischeri."

ERIC Educational Resources Information Center

Slock, James

1995-01-01

Bioluminescence transformation experiments show students the excitement and power of recombinant DNA technology. This laboratory experiment utilizes two plasmids of "Vibrio fischeri" in a transformation experiment. (LZ)

[Evaluation of transfection effectiveness using fluorescein-labelled oligonucleotides and entraster-R siRNA transfection into Plasmodium falciparum].

PubMed

Zhou, Hong-Chang; Gao, Yu-Hui; Shao, Sheng-Wen; Zhang, Hui; Zhang, Ting

2013-12-01

The cultured Plasmodium falciparum parasites were synchronized twice by 5% sorbitol treatment twice (8-hour window), and then incubated at 37 degrees C for 16 h. Parasites were transfected with fluorescein-labelled oligonucleotides (group A) or fluorescein-labelled oligonucleotides+Entranster-R siRNA transfection reagent (group B). After 5 h a part of parasites was evaluated by fluorescence microscopy and flow cytometry. The rest of parasites were washed with RPMI 1640 medium, and then incubated with 500 microl new medium containing 2% fresh erythrocytes for another 12 h, and detected by flow cytometry. The fluorescein-labelled oligonucleotides were localized in erythrocytes in group B, but nearly no fluorescence was observed for group A. Flow cytometry analysis indicated that the transfection efficiency of group B [(47.40 +/- 3.39)%] was higher than that of group A [(0.60 +/- 0.27)%]. In the second cell cycle, the transfection efficiency in group B was (26.85 +/- 2.90)%, while that of group A was nearly zero. The results indicated that Entranster-R siRNA transfection reagent may increase the oligonucleotides transfection efficiency.

Stably stratified canopy flow in complex terrain

NASA Astrophysics Data System (ADS)

Xu, X.; Yi, C.; Kutter, E.

2015-07-01

Stably stratified canopy flow in complex terrain has been considered a difficult condition for measuring net ecosystem-atmosphere exchanges of carbon, water vapor, and energy. A long-standing advection error in eddy-flux measurements is caused by stably stratified canopy flow. Such a condition with strong thermal gradient and less turbulent air is also difficult for modeling. To understand the challenging atmospheric condition for eddy-flux measurements, we use the renormalized group (RNG) k-ϵ turbulence model to investigate the main characteristics of stably stratified canopy flows in complex terrain. In this two-dimensional simulation, we imposed persistent constant heat flux at ground surface and linearly increasing cooling rate in the upper-canopy layer, vertically varying dissipative force from canopy drag elements, buoyancy forcing induced from thermal stratification and the hill terrain. These strong boundary effects keep nonlinearity in the two-dimensional Navier-Stokes equations high enough to generate turbulent behavior. The fundamental characteristics of nighttime canopy flow over complex terrain measured by the small number of available multi-tower advection experiments can be reproduced by this numerical simulation, such as (1) unstable layer in the canopy and super-stable layers associated with flow decoupling in deep canopy and near the top of canopy; (2) sub-canopy drainage flow and drainage flow near the top of canopy in calm night; (3) upward momentum transfer in canopy, downward heat transfer in upper canopy and upward heat transfer in deep canopy; and (4) large buoyancy suppression and weak shear production in strong stability.

NanoLuc: A Small Luciferase is Brightening up the Field of Bioluminescence

PubMed Central

Cai, Weibo

2016-01-01

The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a non-ideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a non-biased manner, allowing the audience to adopt their own views of this novel system. PMID:27045664

The origins of marine bioluminescence: turning oxygen defence mechanisms into deep-sea communication tools.

PubMed

Rees, J F; de Wergifosse, B; Noiset, O; Dubuisson, M; Janssens, B; Thompson, E M

1998-04-01

Bioluminescence, the emission of ecologically functional light by living organisms, emerged independently on several occasions, yet the evolutionary origins of most bioluminescent systems remain obscure. We propose that the luminescent substrates of the luminous reactions (luciferins) are the evolutionary core of most systems, while luciferases, the enzymes catalysing the photogenic oxidation of the luciferin, serve to optimise the expression of the endogenous chemiluminescent properties of the luciferin. Coelenterazine, a luciferin occurring in many marine bioluminescent groups, has strong antioxidative properties as it is highly reactive with reactive oxygen species such as the superoxide anion or peroxides. We suggest that the primary function of coelenterazine was originally the detoxification of the deleterious oxygen derivatives. The functional shift from its antioxidative to its light-emitting function might have occurred when the strength of selection for antioxidative defence mechanisms decreased. This might have been made possible when marine organisms began colonising deeper layers of the oceans, where exposure to oxidative stress is considerably reduced because of reduced light irradiance and lower oxygen levels. A reduction in metabolic activity with increasing depth would also have decreased the endogenous production of reactive oxygen species. Therefore, in these organisms, mechanisms for harnessing the chemiluminescence of coelenterazine in specialised organs could have developed, while the beneficial antioxidative properties were maintained in other tissues. The full range of graded irradiance in the mesopelagic zone, where the majority of organisms are bioluminescent, would have provided a continuum for the selection and improvement of proto-bioluminescence. Although the requirement for oxygen or reactive oxygen species observed in bioluminescent systems reflects the high energy required to produce visible light, it may suggest that oxygen

Marine Bioluminescence: Mechanisms and Evaluation

DTIC Science & Technology

1998-09-30

440 to 506 nm. Ctenophores (41 species) had characteristically longer wavelengths than medusae (34 species), and the wavelengths from siphonophores (25...species) had a bimodal distribution across species. Four species each produced two different wavelengths of light, and in the siphonophore Abylopsis...Bioluminescent spectra of shallow and deep-sea gelatinous zooplankton: medusae, ctenophores and siphonophores . Marine Biology. Haddock, S.H.D., Neilson, D.J

Characterization of an anthraquinone fluor from the bioluminescent, pelagic polychaete Tomopteris

PubMed Central

Francis, Warren R; Powers, Meghan L; Haddock, Steven H D

2014-01-01

Tomopteris is a cosmopolitan genus of polychaetes. Many species produce yellow luminescence in the parapodia when stimulated. Yellow bioluminescence is rare in the ocean, and the components of this luminescent reaction have not been identified. Only a brief description, half a century ago, noted fluorescence in the parapodia with a remarkably similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here, we report the isolation of the fluorescent yellow–orange pigment found in the luminous exudate and in the body of the animals. Liquid chromatography-mass spectrometry revealed the mass to be 270 m/z with a molecular formula of C15H10O5, which ultimately was shown to be aloe-emodin, an anthraquinone previously found in plants. We speculate that aloe-emodin could be a factor for resonant-energy transfer or the oxyluciferin for Tomopteris bioluminescence. PMID:24760626

Study of firefly luciferin oxidation and isomerism as possible inhibition pathways for firefly bioluminescence

NASA Astrophysics Data System (ADS)

Pinto da Silva, Luís; Esteves da Silva, Joaquim C. G.

2014-01-01

Firefly bioluminescence presents a light emitting profile with a form of a flash, due to the firefly luciferase-catalyzed formation of inhibitory products. These impair the binding of the substrate luciferin to the active site of the enzyme. However, this luciferase catalyzed pathways may not be the only ones responsible for the flash profile. The oxidation and isomerisation of the substrate luciferin lead to the formation of compounds that are also known inhibitors of firefly bioluminescence. So, the objective of this Letter was to analyze if these reactions could be capable of interfering with the bioluminescence reaction.

Total evidence phylogeny and the evolution of adult bioluminescence in fireflies (Coleoptera: Lampyridae).

PubMed

Martin, Gavin J; Branham, Marc A; Whiting, Michael F; Bybee, Seth M

2017-02-01

Fireflies are some of the most captivating organisms on the planet. They have a rich history as subjects of scientific study, especially in relation to their bioluminescent behavior. Yet, the phylogenetic relationships of fireflies are still poorly understood. Here, we present the first total evidence approach to reconstruct lampyrid phylogeny using both a molecular matrix from six loci and an extensive morphological matrix. Using this phylogeny we test the hypothesis that adult bioluminescence evolved after the origin of the firefly clade. The ancestral state of adult bioluminescence is recovered as non-bioluminescent with one to six gains and five to ten subsequent losses. The monophyly of the family, as well as the subfamilies is also tested. Ototretinae, Cyphonocerinae, Luciolinae (incl. Pristolycus), Amydetinae, "cheguevarinae" sensu Jeng 2008, and Photurinae are highly supported as monophyletic. With the exception of four taxa, Lampyrinae is also recovered as monophyletic with high support. Based on phylogenetic and morphological data Lamprohiza, Phausis, and Lamprigera are transferred to Lampyridae incertae sedis. Copyright © 2016 Elsevier Inc. All rights reserved.

Calcium phosphate transfection of primary hippocampal neurons.

PubMed

Sun, Miao; Bernard, Laura P; Dibona, Victoria L; Wu, Qian; Zhang, Huaye

2013-11-12

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Bioluminescent bioreporter integrated circuit detection methods

DOEpatents

Simpson, Michael L.; Paulus, Michael J.; Sayler, Gary S.; Applegate, Bruce M.; Ripp, Steven A.

2005-06-14

Disclosed are monolithic bioelectronic devices comprising a bioreporter and an OASIC. These bioluminescent bioreporter integrated circuit are useful in detecting substances such as pollutants, explosives, and heavy-metals residing in inhospitable areas such as groundwater, industrial process vessels, and battlefields. Also disclosed are methods and apparatus for detection of particular analytes, including ammonia and estrogen compounds.

Calcium Phosphate Transfection of Primary Hippocampal Neurons

PubMed Central

DiBona, Victoria L.; Wu, Qian; Zhang, Huaye

2013-01-01

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells. PMID:24300106

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

PubMed

Ausseil, Frederic; Samson, Arnaud; Aussagues, Yannick; Vandenberghe, Isabelle; Creancier, Laurent; Pouny, Isabelle; Kruczynski, Anna; Massiot, Georges; Bailly, Christian

2007-02-01

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

High throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence

PubMed Central

Shultzaberger, Ryan K.; Paddock, Mark L.; Katsuki, Takeo; Greenspan, Ralph J.; Golden, Susan S.

2016-01-01

The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise, and generate meaningful quantitative measurements of clock output levels for advanced analysis. PMID:25662451

Posttranslationally caused bioluminescence burst of the Escherichia coli luciferase reporter strain.

PubMed

Ideguchi, Yamato; Oshikoshi, Yuta; Ryo, Masashi; Motoki, Shogo; Kuwano, Takashi; Tezuka, Takafumi; Aoki, Setsuyuki

2016-01-01

We continuously monitored bioluminescence from a wild-type reporter strain of Escherichia coli (lacp::luc+/WT), which carries the promoter of the lac operon (lacp) fused with the firefly luciferase gene (luc+). This strain showed a bioluminescence burst when shifted into the stationary growth phase. Bioluminescence profiles of other wild-type reporter strains (rpsPp::luc+ and argAp::luc+) and gene-deletion reporter strains (lacp::luc+/crp- and lacp::luc+/lacI-) indicate that transcriptional regulation is not responsible for generation of the burst. Consistently, changes in the luciferase protein levels did not recapitulate the profile of the burst. On the other hand, dissolved oxygen levels increased over the period across the burst, suggesting that the burst is, at least partially, caused by an increase in intracellular oxygen levels. We discuss limits of the firefly luciferase when used as a reporter for gene expression and its potential utility for monitoring metabolic changes in cells.

An optimized expression vector for improving the yield of dengue virus-like particles from transfected insect cells.

PubMed

Charoensri, Nicha; Suphatrakul, Amporn; Sriburi, Rungtawan; Yasanga, Thippawan; Junjhon, Jiraphan; Keelapang, Poonsook; Utaipat, Utaiwan; Puttikhunt, Chunya; Kasinrerk, Watchara; Malasit, Prida; Sittisombut, Nopporn

2014-09-01

Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Orthogonal Luciferase-Luciferin Pairs for Bioluminescence Imaging.

PubMed

Jones, Krysten A; Porterfield, William B; Rathbun, Colin M; McCutcheon, David C; Paley, Miranda A; Prescher, Jennifer A

2017-02-15

Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate "hits" were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.

Increased expression of human leucocyte antigen class I free heavy chains on monocytes of patients with spondyloarthritis and cells transfected with HLA-B27.

PubMed

Ding, Jin; Feng, Yuan; Zheng, Zhao Hui; Li, Xue Yi; Wu, Zhen Biao; Zhu, Ping

2015-02-01

Human leucocyte antigen (HLA)-B27 expression is correlated with spondyloarthritis (SpA), but its role in disease pathogenesis remains unclear. The aim of the study was to determine whether HLA-B27 free heavy chain (FHC) contributes to SpA pathogenesis. Flow cytometry was used to analyse the FHC expression on CD3+ and CD14+ cells in the peripheral blood (PB) and synovial fluid (SF) from SpA patients, healthy controls, and rheumatoid arthritis (RA) patients. Human monocytic U937 cell lines stably expressing enhanced green fluorescence protein (EGFP)/HLA-B27, EGFP/HLA-A2 or EGFP alone were created to further investigate the relation between HLA-B27 and FHC expression. The relative FHC level on CD14+ PB cells was significantly higher in SpA patients than in controls, but lower than on the SF cells of SpA patients. No significant correlation was found for relative FHC expression with HLA-B27 or β2-microglobulin expression. HLA-B27-transfected U937 cells expressed higher FHC levels than either EGFP/HLA-A2- or EGFP-transfected cells. HLA class I FHC expression was significantly increased on monocytes of SpA patients and HLA-B27-transfected cells, implying that FHC, perhaps mostly derived from HLA-B27, plays an important role in SpA pathogenesis. © 2014 John Wiley & Sons Ltd.

Bioluminescent indicators for Ca2+ based on split Renilla luciferase complementation in living cells.

PubMed

Kaihara, Asami; Umezawa, Yoshio; Furukawa, Tetsushi

2008-01-01

Genetically encoded bioluminescent indicators for intracellular Ca2+ are described here with CaM-M13 interaction-induced complementation of split Renilla luciferase. The Ca2+-induced interaction between CaM and M13 leads to complementation of the N- and C-terminal halves of split Renilla luciferase in living cells. This intramolecular interaction results in the spontaneous and simultaneous emission of bioluminescence split Renilla luciferase. This is how intracellular Ca2+ is illuminated with the intramolecular complementation of split Renilla luciferase. The Ca2+-dependent spontaneous and simultaneous emission of bioluminescence promises to reveal Ca2+ dynamics in living cells, and also in vivo using the present indicators.

Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

NASA Astrophysics Data System (ADS)

Close, Dan; Webb, James; Ripp, Steven; Patterson, Stacey; Sayler, Gary

2012-06-01

Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

[Development of a mouse cell line containing stably integrated copies of pMCLacI/Neo plasmid: a model for studying mutations in vitro].

PubMed

Lu, Y; Li, H; Fu, J

2000-04-01

To establish a suitable model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells. The NIH3T3 cells were transfected with the linearized pMCLacI/Neo DNAs by liposome-mediated transfection, and grew in the presence of G418. One drug resistant cell clone was selected to proliferate and to be analyzed with Southern blot and RT-PCR analyses on its genomic DNAs. (1) Multiple copies of pMCLacI/Neo plasmid DNA were intactly integrated in the genomic DNAs of the cell clone. (2) One of lac I target genes in the integrated plasmid could be transcribed in the NIH3T3 cells while the other could not. (3) The pMCLacI/Neo plasmid DNA could be efficiently rescued from the genomic DNAs of the cell clone with the average rescue efficiency of 410 cfu/microg DNA. The NIH3T3 cell line containing copies of a stably integrated pMCLacI/Neo has been established. The two lacI target genes in the cell line could imitate the functional states of expressed and non-expressed genes in mammalian cells respectively. The cell line will be a useful model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells.

PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.

PubMed

Inada, Emi; Saitoh, Issei; Watanabe, Satoshi; Aoki, Reiji; Miura, Hiromi; Ohtsuka, Masato; Murakami, Tomoya; Sawami, Tadashi; Yamasaki, Youichi; Sato, Masahiro

2015-09-14

The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.

Expression of intracellular interferon-alpha confers antiviral properties in transfected bovine fetal fibroblasts and does not affect the full development of SCNT embryos.

PubMed

Yu, Dawei; Zhang, Shoufeng; Du, Weihua; Zhang, Jinxia; Fan, Zongxing; Hao, Haisheng; Liu, Yan; Zhao, Xueming; Qin, Tong; Zhu, Huabin

2014-01-01

Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α) (without secretory signal sequence) gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT). Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9%) became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR) and the 2'-5'-oligoadenylate synthetase gene (2'-5' OAS), which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2'-5' OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.

Hyperspectral and multispectral bioluminescence optical tomography for small animal imaging.

PubMed

Chaudhari, Abhijit J; Darvas, Felix; Bading, James R; Moats, Rex A; Conti, Peter S; Smith, Desmond J; Cherry, Simon R; Leahy, Richard M

2005-12-07

For bioluminescence imaging studies in small animals, it is important to be able to accurately localize the three-dimensional (3D) distribution of the underlying bioluminescent source. The spectrum of light produced by the source that escapes the subject varies with the depth of the emission source because of the wavelength-dependence of the optical properties of tissue. Consequently, multispectral or hyperspectral data acquisition should help in the 3D localization of deep sources. In this paper, we describe a framework for fully 3D bioluminescence tomographic image acquisition and reconstruction that exploits spectral information. We describe regularized tomographic reconstruction techniques that use semi-infinite slab or FEM-based diffusion approximations of photon transport through turbid media. Singular value decomposition analysis was used for data dimensionality reduction and to illustrate the advantage of using hyperspectral rather than achromatic data. Simulation studies in an atlas-mouse geometry indicated that sub-millimeter resolution may be attainable given accurate knowledge of the optical properties of the animal. A fixed arrangement of mirrors and a single CCD camera were used for simultaneous acquisition of multispectral imaging data over most of the surface of the animal. Phantom studies conducted using this system demonstrated our ability to accurately localize deep point-like sources and show that a resolution of 1.5 to 2.2 mm for depths up to 6 mm can be achieved. We also include an in vivo study of a mouse with a brain tumour expressing firefly luciferase. Co-registration of the reconstructed 3D bioluminescent image with magnetic resonance images indicated good anatomical localization of the tumour.

Continuous, real-time bioimaging of chemical bioavailability and toxicology using autonomously bioluminescent human cell lines

NASA Astrophysics Data System (ADS)

Xu, Tingting; Close, Dan M.; Webb, James D.; Price, Sarah L.; Ripp, Steven A.; Sayler, Gary S.

2013-05-01

Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety.

Modeling and image reconstruction in spectrally resolved bioluminescence tomography

NASA Astrophysics Data System (ADS)

Dehghani, Hamid; Pogue, Brian W.; Davis, Scott C.; Patterson, Michael S.

2007-02-01

Recent interest in modeling and reconstruction algorithms for Bioluminescence Tomography (BLT) has increased and led to the general consensus that non-spectrally resolved intensity-based BLT results in a non-unique problem. However, the light emitted from, for example firefly Luciferase, is widely distributed over the band of wavelengths from 500 nm to 650 nm and above, with the dominant fraction emitted from tissue being above 550 nm. This paper demonstrates the development of an algorithm used for multi-wavelength 3D spectrally resolved BLT image reconstruction in a mouse model. It is shown that using a single view data, bioluminescence sources of up to 15 mm deep can be successfully recovered given correct information about the underlying tissue absorption and scatter.

Caged ATP - an internal calibration method for ATP bioluminescence assays.

PubMed

Calvert, R M; Hopkins, H C; Reilly, M J; Forsythe, S J

2000-03-01

ATP bioluminescence, based on the firefly luciferase system, is used for the rapid determination of hygienic practices in the food industry. This study has demonstrated the use of caged ATP as an internal ATP standard and quantified the effects of industrial cleansing solutions, alcoholic beverages and pH on firefly luciferase activity. The light signal was quenched 6-47% by five cleansing solutions at standard working concentrations. Ethanol at 1% (v/v) inhibited bioluminescence by 15% (w/v) whereas concentrations above 4% enhanced the light output. The light signal was quenched by 20-25% at pH values below pH 4 and above pH 10.

Bacterial bioluminescence response to long-term exposure to reverse osmosis treated effluents from dye industries.

PubMed

Ravindran, J; Manikandan, B; Shirodkar, P V; Francis, K X; Mani Murali, R; Vethamony, P

2014-10-01

The bacterial bioluminescence assay is one of the novel means for toxicity detection. The bioluminescence response of 2 marine bioluminescent bacteria was tested upon their long-term exposure to 9 different reverse osmosis (RO) rejects with varying chemical composition sampled from various dye industries. Bioluminescent bacteria were cultured in the RO reject samples, at different concentrations, and their growth rate and luminescence was measured for 24 h. The RO reject samples caused sublethal effects upon exposure and retarded the growth of bacteria, confirming their toxic nature. Further, continuation of the exposure showed that the initial luminescence, though reduced, recovered and increased beyond the control cultures irrespective of cell density, and finally decreased once again. The present study emphasizes the need of evolving a long-term exposure assay and shows that the method followed in this study is suitable to evaluate the toxicants that exert delayed toxicity, using lower concentrations of toxicants as well as coloured samples.

Image analyzing method to evaluate in situ bioluminescence from an obligate anaerobe cultivated under various dissolved oxygen concentrations.

PubMed

Ninomiya, Kazuaki; Yamada, Ryuji; Matsumoto, Masami; Fukiya, Satoru; Katayama, Takane; Ogino, Chiaki; Shimizu, Nobuaki

2013-02-01

An image analyzing method was developed to evaluate in situ bioluminescence expression, without exposing the culture sample to the ambient oxygen atmosphere. Using this method, we investigated the effect of dissolved oxygen concentration on bioluminescence from an obligate anaerobe Bifidobacterium longum expressing bacterial luciferase which catalyzes an oxygen-requiring bioluminescent reaction. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

An adaptive regularization parameter choice strategy for multispectral bioluminescence tomography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng Jinchao; Qin Chenghu; Jia Kebin

2011-11-15

Purpose: Bioluminescence tomography (BLT) provides an effective tool for monitoring physiological and pathological activities in vivo. However, the measured data in bioluminescence imaging are corrupted by noise. Therefore, regularization methods are commonly used to find a regularized solution. Nevertheless, for the quality of the reconstructed bioluminescent source obtained by regularization methods, the choice of the regularization parameters is crucial. To date, the selection of regularization parameters remains challenging. With regards to the above problems, the authors proposed a BLT reconstruction algorithm with an adaptive parameter choice rule. Methods: The proposed reconstruction algorithm uses a diffusion equation for modeling the bioluminescentmore » photon transport. The diffusion equation is solved with a finite element method. Computed tomography (CT) images provide anatomical information regarding the geometry of the small animal and its internal organs. To reduce the ill-posedness of BLT, spectral information and the optimal permissible source region are employed. Then, the relationship between the unknown source distribution and multiview and multispectral boundary measurements is established based on the finite element method and the optimal permissible source region. Since the measured data are noisy, the BLT reconstruction is formulated as l{sub 2} data fidelity and a general regularization term. When choosing the regularization parameters for BLT, an efficient model function approach is proposed, which does not require knowledge of the noise level. This approach only requests the computation of the residual and regularized solution norm. With this knowledge, we construct the model function to approximate the objective function, and the regularization parameter is updated iteratively. Results: First, the micro-CT based mouse phantom was used for simulation verification. Simulation experiments were used to illustrate why multispectral data

Production and characterization of active recombinant interleukin-12/eGFP fusion protein in stably-transfected DF1 chicken cells.

PubMed

Wu, Hsing Chieh; Chen, Yu San; Shen, Pin Chun; Shien, Jui Hung; Lee, Long Huw; Chiu, Hua Hsien

2015-01-01

The adjuvant activity of chicken interleukin-12 (chIL-12) protein has been described as similar to that of mammalian IL-12. Recombinant chIL-12 can be produced using several methods, but chIL-12 production in eukaryotic cells is lower than that in prokaryotic cells. Stimulating compounds, such as dimethyl sulfoxide (DMSO), can be added to animal cell cultures to overcome this drawback. In this study, we constructed a cell line, DF1/chIL-12 which stably expressed a fusion protein, chIL-12 and enhanced green fluorescent protein (eGFP) connected by a (G4 S)3 linker sequence. Fusion protein production was increased when cells were cultured in the presence of DMSO. When 1 × 10(6) DF1/chIL-12 cells were inoculated in a T-175 flask containing 30 mL of media, incubated for 15 h, and further cultivated in the presence of 4% DMSO for 48 h, the production of total fusion protein was mostly enhanced compared with the production of total fusion protein by using cell lysates induced with DMSO at other concentrations. The concentrations of the unpurified and purified total fusion proteins in cell lysates were 2,781 ± 2.72 ng mL(-1) and 2,207 ± 3.28 ng mL(-1) , respectively. The recovery rate was 79%. The fusion protein stimulated chicken splenocytes to produce IFN-γ, which was measured using an enzyme-linked immunosorbent assay, in the culture supernatant, indicating that treating DF1/chIL-12 cells with DMSO or producing chIL-12 in a fusion protein form does not have adverse effects on the bioactivity of chIL-12. © 2015 American Institute of Chemical Engineers.

Detection of organic compounds with whole-cell bioluminescent bioassays.

PubMed

Xu, Tingting; Close, Dan; Smartt, Abby; Ripp, Steven; Sayler, Gary

2014-01-01

Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices.

Three-dimensional light-tissue interaction models for bioluminescence tomography

NASA Astrophysics Data System (ADS)

Côté, D.; Allard, M.; Henkelman, R. M.; Vitkin, I. A.

2005-09-01

Many diagnostic and therapeutic approaches in medical physics today take advantage of the unique properties of light and its interaction with tissues. Because light scatters in tissue, our ability to develop these techniques depends critically on our knowledge of the distribution of light in tissue. Solutions to the diffusion equation can provide such information, but often lack the flexibility required for more general problems that involve, for instance, inhomogeneous optical properties, light polarization, arbitrary three-dimensional geometries, or arbitrary scattering. Monte Carlo techniques, which statistically sample the light distribution in tissue, offer a better alternative to analytical models. First, we discuss our implementation of a validated three-dimensional polarization-sensitive Monte Carlo algorithm and demonstrate its generality with respect to the geometry and scattering models it can treat. Second, we apply our model to bioluminescence tomography. After appropriate genetic modifications to cell lines, bioluminescence can be used as an indicator of cell activity, and is often used to study tumour growth and treatment in animal models. However, the amount of light escaping the animal is strongly dependent on the position and size of the tumour. Using forward models and structural data from magnetic resonance imaging, we show how the models can help to determine the location and size of tumour made of bioluminescent cancer cells in the brain of a mouse.

Detection of Organic Compounds with Whole-Cell Bioluminescent Bioassays

PubMed Central

Xu, Tingting; Close, Dan; Smartt, Abby; Ripp, Steven

2015-01-01

Natural and manmade organic chemicals are widely deposited across a diverse range of ecosystems including air, surface water, groundwater, wastewater, soil, sediment, and marine environments. Some organic compounds, despite their industrial values, are toxic to living organisms and pose significant health risks to humans and wildlife. Detection and monitoring of these organic pollutants in environmental matrices therefore is of great interest and need for remediation and health risk assessment. Although these detections have traditionally been performed using analytical chemical approaches that offer highly sensitive and specific identification of target compounds, these methods require specialized equipment and trained operators, and fail to describe potential bioavailable effects on living organisms. Alternatively, the integration of bioluminescent systems into whole-cell bioreporters presents a new capacity for organic compound detection. These bioreporters are constructed by incorporating reporter genes into catabolic or signaling pathways that are present within living cells and emit a bioluminescent signal that can be detected upon exposure to target chemicals. Although relatively less specific compared to analytical methods, bioluminescent bioassays are more cost-effective, more rapid, can be scaled to higher throughput, and can be designed to report not only the presence but also the bioavailability of target substances. This chapter reviews available bacterial and eukaryotic whole-cell bioreporters for sensing organic pollutants and their applications in a variety of sample matrices. PMID:25084996

Smartphone-based low light detection for bioluminescence application

USDA-ARS?s Scientific Manuscript database

We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation ...

Monitoring Neural Activity with Bioluminescence during Natural Behavior

PubMed Central

Naumann, Eva A.; Kampff, Adam R.; Prober, David A.; Schier, Alexander F.; Engert, Florian

2010-01-01

Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. Here we describe the use of bioluminescence to non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish expressing the Ca2+-sensitive photoprotein GFP-apoAequorin (GA) in most neurons generated large and fast bioluminescent signals related to neural activity, neuroluminescence, that could be recorded continuously for many days. To test the limits of this technique, GA was specifically targeted to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of ~20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Thus, our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior. PMID:20305645

Liquid-chromatographic separation and on-line bioluminescence detection of creatine kinase isoenzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

1980-01-01

Isoenzymes of creatine kinase were separated by anion-exchange chromatography, with use of an elution gradient containing lithium acetate (0.1 to 0.6 mol/L). A stream splitter was used to divert a 5% side stream of column effluent, which was subsequently mixed with the reagents necessary for bioluminescence assay of the separated isoenzymes. The use of the stream splitter greatly decreased the rate of consumption of reagent and, when combined with a peristaltic pumping system, permitted independent control of the side-stream flow rate. Thus both the residence interval in a delay coil in which the ATP reaction product is formed and themore » bioluminescence emission was monitored in a flow-through fluorometer without use of an external light source or filters. Separation and detection of the isoenzymes of creatine kinase were rapid, sensitive, and highly selective. The incremental decrease of bioluminescence response owing to inhibition by the ions in the eluent was less than 31% across the entire gradient.« less

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil.

PubMed

Amaral, D T; Arnoldi, F G C; Rosa, S P; Viviani, V R

2014-08-01

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.

Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

PubMed

Falagan-Lotsch, Priscila; Lopes, Talíria Silva; Ferreira, Nívea; Balthazar, Nathália; Monteiro, Antônio M; Borojevic, Radovan; Granjeiro, José Mauro

2015-11-01

Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed. Copyright © 2015 Elsevier B.V. All rights

Species abundance, sexual encounter and bioluminescent signalling in the deep sea.

PubMed Central

Herring, P J

2000-01-01

The problems faced by deep-sea animals in achieving sexual and other encounters require sensory and effector systems the synergy of which can span the often very substantial distances that separate individuals. Bioluminescent systems provide one of the links between individuals, and the sexual dimorphism of some photophores suggests that they are employed to attract a mate. However, nearest-neighbour values for many deep-sea animals put them beyond the effective range of bioluminescent signals and it is therefore likely that these signals are employed at intermediate ranges, once an initial contact (perhaps olfactory) has been made. PMID:11079413

A bright future for bioluminescent imaging in viral research

PubMed Central

Coleman, Stewart M; McGregor, Alistair

2015-01-01

Summary Bioluminescence imaging (BLI) has emerged as a powerful tool in the study of animal models of viral disease. BLI enables real-time in vivo study of viral infection, host immune response and the efficacy of intervention strategies. Substrate dependent light emitting luciferase enzyme when incorporated into a virus as a reporter gene enables detection of bioluminescence from infected cells using sensitive charge-coupled device (CCD) camera systems. Advantages of BLI include low background, real-time tracking of infection in the same animal and reduction in the requirement for larger animal numbers. Transgenic luciferase-tagged mice enable the use of pre-existing nontagged viruses in BLI studies. Continued development in luciferase reporter genes, substrates, transgenic animals and imaging systems will greatly enhance future BLI strategies in viral research. PMID:26413138

Seasonal Changes of Bioluminescence in Photosynthetic and Heterotrophic Dinoflagellates at San Clemente Island

DTIC Science & Technology

2012-02-01

were calculated using n-2 degrees of freedom. 2.2 Nutrient and chlorophyll data Nitrate and Chl a levels were obtained from archived CalCOFI data...a later increase in photosynthetic biomass in the fall. However, Chl a levels were actually low during the period when bioluminescence was high and...for 1994-1995 which may suggest that as Chl a levels increased, so did bioluminescence cell-1 (Figure 7c). These field measurements support previous

Bioluminescent magnetic nanoparticles as potential imaging agents for mammalian spermatozoa.

PubMed

Vasquez, Erick S; Feugang, Jean M; Willard, Scott T; Ryan, Peter L; Walters, Keisha B

2016-03-17

Nanoparticles have emerged as key materials for developing applications in nanomedicine, nanobiotechnology, bioimaging and theranostics. Existing bioimaging technologies include bioluminescent resonance energy transfer-conjugated quantum dots (BRET-QDs). Despite the current use of BRET-QDs for bioimaging, there are strong concerns about QD nanocomposites containing cadmium which exhibits potential cellular toxicity. In this study, bioluminescent composites comprised of magnetic nanoparticles and firefly luciferase (Photinus pyralis) are examined as potential light-emitting agents for imaging, detection, and tracking mammalian spermatozoa. Characterization was carried out using infrared spectroscopy, TEM and cryo-TEM imaging, and ζ-potential measurements to demonstrate the successful preparation of these nanocomposites. Binding interactions between the synthesized nanoparticles and spermatozoon were characterized using confocal and atomic/magnetic force microscopy. Bioluminescence imaging and UV-visible-NIR microscopy results showed light emission from sperm samples incubated with the firefly luciferase-modified nanoparticles. Therefore, these newly synthesized luciferase-modified magnetic nanoparticles show promise as substitutes for QD labeling, and can potentially also be used for in vivo manipulation and tracking, as well as MRI techniques. These preliminary data indicate that luciferase-magnetic nanoparticle composites can potentially be used for spermatozoa detection and imaging. Their magnetic properties add additional functionality to allow for manipulation, sorting, or tracking of cells using magnetic techniques.

Bacteria as part of bioluminescence emission at the deep ANTARES station (North-Western Mediterranean Sea) during a one-year survey

NASA Astrophysics Data System (ADS)

Martini, S.; Michotey, V.; Casalot, L.; Bonin, P.; Guasco, S.; Garel, M.; Tamburini, C.

2016-10-01

Bioluminescent bacteria have been studied during a one-year survey in 2011 at the deep ANTARES site (Northwestern Mediterranean Sea, 2000 m depth). The neutrino underwater telescope ANTARES, located at this station, has been used to record the bioluminescence at the same depth. Together with these data, environmental variables (potential temperature, salinity, nutrients, dissolved organic carbon and oxygen) have been characterized in water samples. The year 2011 was characterized by relatively stable conditions, as revealed by minor variability in the monitored oceanographic variables, by low bioluminescence and low current speed. This suggests weak eukaryote participation and mainly non-stimulated light emission. Hence, no processes of dense water have affected the ANTARES station during this survey. Abundance of bioluminescent bacteria belonging to Photobacterium genus, measured by qPCR of the luxF gene, ranged from 1.4×102 to 7.2×102 genes mL-1. Their effective activity was confirmed through mRNA luxF quantification. Our results reveal that bioluminescent bacteria appeared more active than the total counterpart of bacteria, suggesting an ecological benefit of this feature such as favoring interaction with macro-organisms. Moreover, these results show that part of the bioluminescence, recorded at 2000 m depth over one year, could be due to bioluminescent bacteria in stable hydrological conditions.

Bioluminescence enhancement through an added washing protocol enabling a greater sensitivity to carbofuran toxicity.

PubMed

Jia, Kun; Eltzov, Evgeni; Marks, Robert S; Ionescu, Rodica E

2013-10-01

The effects of carbofuran toxicity on a genetically modified bacterial strain E. coli DPD2794 were enhanced using a new bioluminescent protocol which consisted of three consecutive steps: incubation, washing and luminescence reading. Specifically, in the first step, several concentrations of carbofuran aqueous solutions were incubated with different bacterial suspensions at recorded optical densities for different lengths of time. Thereafter, the resulting bacterial/toxicant mixtures were centrifuged and the aged cellular supernatant replaced with fresh medium. In the final step, the carbofuran- induced bioluminescence to the exposed E. coli DPD2794 bacteria was shown to provide a faster and higher intensity when recorded at a higher temperature at30°C which is not usually used in the literature. It was found that the incubation time and the replacement of aged cellular medium were essential factors to distinguish different concentrations of carbofuran in the bioluminescent assays. From our results, the optimum incubation time for a "light ON" bioluminescence detection of the effect of carbofuran was 6h. Thanks to the replacement of the aged cellular medium, a group of additional peaks starting around 30min were observed and we used the corresponding areas under the curve (AUC) at different contents of carbofuran to produce the calibration curve. Based on the new protocol, a carbofuran concentration of 0.5pg/mL can be easily determined in a microtiter plate bioluminescent assay, while a non-wash protocol provides an unexplainable order of curve evolutionswhich does not allow the user to determine the concentration. Copyright © 2013 Elsevier Inc. All rights reserved.

Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA.

PubMed

Hurrelbrink, R J; Nestorowicz, A; McMinn, P C

1999-12-01

An infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was constructed by stably inserting genome-length cDNA into the low-copy-number plasmid vector pMC18. Designated pMVE-1-51, the clone consisted of genome-length cDNA of MVE-1-51 under the control of a T7 RNA polymerase promoter. The clone was constructed by using existing components of a cDNA library, in addition to cDNA of the 3' terminus derived by RT-PCR of poly(A)-tailed viral RNA. Upon comparison with other flavivirus sequences, the previously undetermined sequence of the 3' UTR was found to contain elements conserved throughout the genus FLAVIVIRUS: RNA transcribed from pMVE-1-51 and subsequently transfected into BHK-21 cells generated infectious virus. The plaque morphology, replication kinetics and antigenic profile of clone-derived virus (CDV-1-51) was similar to the parental virus in vitro. Furthermore, the virulence properties of CDV-1-51 and MVE-1-51 (LD(50) values and mortality profiles) were found to be identical in vivo in the mouse model. Through site-directed mutagenesis, the infectious clone should serve as a valuable tool for investigating the molecular determinants of virulence in MVE virus.

Enhancement and optimization of plasmid expression in femtosecond optical transfection.

PubMed

Praveen, Bavishna B; Stevenson, David J; Antkowiak, Maciej; Dholakia, Kishan; Gunn-Moore, Frank J

2011-04-01

Cell transfection using femtosecond lasers is gaining importance for its proven ability to achieve selective transfection in a sterile and relatively non-invasive manner. However, the net efficiency of this technique is limited due to a number of factors that ultimately makes it difficult to be used as a viable and widely used technique. We report here a method to achieve significant enhancement in the efficiency of femtosecond optical transfection. The transfection procedure is modified by incorporating a suitable synthetic peptide containing nuclear localization and DNA binding sequences, assisting DNA import into the nucleus. We achieved a 3-fold enhancement in the transfection efficiency for adherent Chinese Hamster Ovary (CHO-K1) cells with this modified protocol. Further, in the presence of this biochemical reagent, we were able to reduce the required plasmid concentration by ~70% without compromising the transfection efficiency. Also, we report for the first time the successful photo-transfection of recently trypsinised cells with significantly high transfection efficiency when transfected with modified plasmid. This paves the way for the development of high throughput microfluidic optical transfection devices. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effect of environmental pH on polymeric transfection efficiency.

PubMed

Kang, Han Chang; Samsonova, Olga; Kang, Sun-Woong; Bae, You Han

2012-02-01

Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6-7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1-2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers. Copyright © 2011 Elsevier Ltd. All rights reserved.

Bioluminescence for determining energy state of plants

NASA Technical Reports Server (NTRS)

Ching, T. M.

1975-01-01

Bioluminescence produced by the luciferin-luciferase system is a very sensitive assay for ATP content in extracts of plant materials. The ATP test for seed and pollen viability and vigor is presented, along with prediction of high growth potential and productivity in new crosses and selections of breeding materials. ATP as an indicator for environmental quality, stresses, and metabolic regulation is also considered.

Neuronal network imaging in acute slices using Ca2+ sensitive bioluminescent reporter.

PubMed

Tricoire, Ludovic; Lambolez, Bertrand

2014-01-01

Genetically encoded indicators are valuable tools to study intracellular signaling cascades in real time using fluorescent or bioluminescent imaging techniques. Imaging of Ca(2+) indicators is widely used to record transient intracellular Ca(2+) increases associated with bioelectrical activity. The natural bioluminescent Ca(2+) sensor aequorin has been historically the first Ca(2+) indicator used to address biological questions. Aequorin imaging offers several advantages over fluorescent reporters: it is virtually devoid of background signal; it does not require light excitation and interferes little with intracellular processes. Genetically encoded sensors such as aequorin are commonly used in dissociated cultured cells; however it becomes more challenging to express them in differentiated intact specimen such as brain tissue. Here we describe a method to express a GFP-aequorin (GA) fusion protein in pyramidal cells of neocortical acute slices using recombinant Sindbis virus. This technique allows expressing GA in several hundreds of neurons on the same slice and to perform the bioluminescence recording of Ca(2+) transients in single neurons or multiple neurons simultaneously.

A human GRPr-transfected Ace-1 canine prostate cancer model in mice.

PubMed

Ding, Haiming; Kothandaraman, Shankaran; Gong, Li; Williams, Michelle M; Dirksen, Wessel P; Rosol, Thomas J; Tweedle, Michael F

2016-06-01

A versatile drug screening system was developed to simplify early targeted drug discovery in mice and then translate readily from mice to a dog prostate cancer model that more fully replicates the features of human prostate cancer. We stably transfected human cDNA of the GRPr bombesin (BBN) receptor subtype to canine Ace-1 prostate cancer cells (Ace-1(huGRPr) ). Expression was examined by (125) I-Tyr(4) -BBN competition, calcium stimulation assay, and fluorescent microscopy. A dual tumor nude mouse xenograft model was developed from Ace-1(CMV) (vector transfected Ace-1) and Ace-1(huGRPr) cells. The model was used to explore the in vivo behavior of two new IRDye800-labeled GRPr binding optical imaging agents: 800-G-Abz4-t-BBN, from a GRPr agonist peptide, and 800-G-Abz4-STAT, from a GRPr antagonist peptide, by imaging the tumor mice and dissected organs. Both agents bound Ace-1(huGRPr) and PC-3, a known GRPr-expressing human prostate cancer cell line, with 4-13 nM IC50 against (125) I-Tyr(4) -BBN, but did not bind Ace-1(CMV) cells (vector transfected). Binding was blocked by bombesin. Ca(2+) activation assays demonstrated that Ace-1(huGPRr) expressed biologically active GRPr. Both Ace-1 cell lines grew in the flanks of 100% of the nude mice and formed tumors of ∼0.5 cm diameter in 1 week. In vivo imaging of the mice at 800 nm emission showed GRPr+: GRPr- tumor signal brighter by a factor of two at 24 h post IV administration of 10 nmol of the imaging agents. Blood retention (4-8% ID at 1 h) was greater by a factor >10 and cumulative urine accumulation (28-30% at 4 h) was less by a factor 2 compared to a radioactive analog of the t-BBN containing agent, (177) LuAMBA, probably due to binding to blood albumin, which we confirmed in a mouse serum assay. The dual tumor Ace-1(CMV) /Ace-1(huGRPr) model system provides a rapid test of specific to nonspecific binding of new GRPr avid agents in a model that will extend logically to the known Ace-1 orthotopic

Monitoring Bloom Dynamics of a Common Coastal Bioluminescent Ctenophore

DTIC Science & Technology

2010-09-30

photodiodes. IMPACT/APPLICATIONS More frequent and more rapidly developing jellyfish blooms, especially Mnemiopsis leidyi as well as Harmful Algal...To meet the need for a bioluminescent jellyfish monitoring and forecasting system, predictive models will depend upon dense networks of sensor

Modeling the Conducting Stably-Stratified Layer of the Earth's Core

NASA Astrophysics Data System (ADS)

Petitdemange, L.; Philidet, J.; Gissinger, C.

2017-12-01

Observations of the Earth magnetic field as well as recent theoretical works tend to show that the Earth's outer liquid core is mostly comprised of a convective zone in which the Earth's magnetic field is generated - likely by dynamo action -, but also features a thin, stably stratified layer at the top of the core.We carry out direct numerical simulations by modeling this thin layer as an axisymmetric spherical Couette flow for a stably stratified fluid embedded in a dipolar magnetic field. The dynamo region is modeled by a conducting inner core rotating slightly faster than the insulating mantle due to magnetic torques acting on it, such that a weak differential rotation (low Rossby limit) can develop in the stably stratified layer.In the case of a non-stratified fluid, the combined action of the differential rotation and the magnetic field leads to the well known regime of `super-rotation', in which the fluid rotates faster than the inner core. Whereas in the classical case, this super-rotation is known to vanish in the magnetostrophic limit, we show here that the fluid stratification significantly extends the magnitude of the super-rotation, keeping this phenomenon relevant for the Earth core. Finally, we study how the shear layers generated by this new state might give birth to magnetohydrodynamic instabilities or waves impacting the secular variations or jerks of the Earth's magnetic field.

Bacterial bioluminescence onset and quenching: a dynamical model for a quorum sensing-mediated property

PubMed Central

Side, Domenico Delle; Nassisi, Vincenzo; Pennetta, Cecilia; Alifano, Pietro; Di Salvo, Marco; Talà, Adelfia; Chechkin, Aleksei; Seno, Flavio

2017-01-01

We present an effective dynamical model for the onset of bacterial bioluminescence, one of the most studied quorum sensing-mediated traits. Our model is built upon simple equations that describe the growth of the bacterial colony, the production and accumulation of autoinducer signal molecules, their sensing within bacterial cells, and the ensuing quorum activation mechanism that triggers bioluminescent emission. The model is directly tested to quantitatively reproduce the experimental distributions of photon emission times, previously measured for bacterial colonies of Vibrio jasicida, a luminescent bacterium belonging to the Harveyi clade, growing in a highly drying environment. A distinctive and novel feature of the proposed model is bioluminescence ‘quenching’ after a given time elapsed from activation. Using an advanced fitting procedure based on the simulated annealing algorithm, we are able to infer from the experimental observations the biochemical parameters used in the model. Such parameters are in good agreement with the literature data. As a further result, we find that, at least in our experimental conditions, light emission in bioluminescent bacteria appears to originate from a subtle balance between colony growth and quorum activation due to autoinducers diffusion, with the two phenomena occurring on the same time scale. This finding is consistent with a negative feedback mechanism previously reported for Vibrio harveyi. PMID:29308273

Bacterial bioluminescence onset and quenching: a dynamical model for a quorum sensing-mediated property.

PubMed

Side, Domenico Delle; Nassisi, Vincenzo; Pennetta, Cecilia; Alifano, Pietro; Di Salvo, Marco; Talà, Adelfia; Chechkin, Aleksei; Seno, Flavio; Trovato, Antonio

2017-12-01

We present an effective dynamical model for the onset of bacterial bioluminescence, one of the most studied quorum sensing-mediated traits. Our model is built upon simple equations that describe the growth of the bacterial colony, the production and accumulation of autoinducer signal molecules, their sensing within bacterial cells, and the ensuing quorum activation mechanism that triggers bioluminescent emission. The model is directly tested to quantitatively reproduce the experimental distributions of photon emission times, previously measured for bacterial colonies of Vibrio jasicida , a luminescent bacterium belonging to the Harveyi clade, growing in a highly drying environment. A distinctive and novel feature of the proposed model is bioluminescence 'quenching' after a given time elapsed from activation. Using an advanced fitting procedure based on the simulated annealing algorithm, we are able to infer from the experimental observations the biochemical parameters used in the model. Such parameters are in good agreement with the literature data. As a further result, we find that, at least in our experimental conditions, light emission in bioluminescent bacteria appears to originate from a subtle balance between colony growth and quorum activation due to autoinducers diffusion, with the two phenomena occurring on the same time scale. This finding is consistent with a negative feedback mechanism previously reported for Vibrio harveyi .

Deep-sea bioluminescence blooms after dense water formation at the ocean surface.

PubMed

Tamburini, Christian; Canals, Miquel; Durrieu de Madron, Xavier; Houpert, Loïc; Lefèvre, Dominique; Martini, Séverine; D'Ortenzio, Fabrizio; Robert, Anne; Testor, Pierre; Aguilar, Juan Antonio; Samarai, Imen Al; Albert, Arnaud; André, Michel; Anghinolfi, Marco; Anton, Gisela; Anvar, Shebli; Ardid, Miguel; Jesus, Ana Carolina Assis; Astraatmadja, Tri L; Aubert, Jean-Jacques; Baret, Bruny; Basa, Stéphane; Bertin, Vincent; Biagi, Simone; Bigi, Armando; Bigongiari, Ciro; Bogazzi, Claudio; Bou-Cabo, Manuel; Bouhou, Boutayeb; Bouwhuis, Mieke C; Brunner, Jurgen; Busto, José; Camarena, Francisco; Capone, Antonio; Cârloganu, Christina; Carminati, Giada; Carr, John; Cecchini, Stefano; Charif, Ziad; Charvis, Philippe; Chiarusi, Tommaso; Circella, Marco; Coniglione, Rosa; Costantini, Heide; Coyle, Paschal; Curtil, Christian; Decowski, Patrick; Dekeyser, Ivan; Deschamps, Anne; Donzaud, Corinne; Dornic, Damien; Dorosti, Hasankiadeh Q; Drouhin, Doriane; Eberl, Thomas; Emanuele, Umberto; Ernenwein, Jean-Pierre; Escoffier, Stéphanie; Fermani, Paolo; Ferri, Marcelino; Flaminio, Vincenzo; Folger, Florian; Fritsch, Ulf; Fuda, Jean-Luc; Galatà, Salvatore; Gay, Pascal; Giacomelli, Giorgio; Giordano, Valentina; Gómez-González, Juan-Pablo; Graf, Kay; Guillard, Goulven; Halladjian, Garadeb; Hallewell, Gregory; van Haren, Hans; Hartman, Joris; Heijboer, Aart J; Hello, Yann; Hernández-Rey, Juan Jose; Herold, Bjoern; Hößl, Jurgen; Hsu, Ching-Cheng; de Jong, Marteen; Kadler, Matthias; Kalekin, Oleg; Kappes, Alexander; Katz, Uli; Kavatsyuk, Oksana; Kooijman, Paul; Kopper, Claudio; Kouchner, Antoine; Kreykenbohm, Ingo; Kulikovskiy, Vladimir; Lahmann, Robert; Lamare, Patrick; Larosa, Giuseppina; Lattuada, Dario; Lim, Gordon; Presti, Domenico Lo; Loehner, Herbert; Loucatos, Sotiris; Mangano, Salvatore; Marcelin, Michel; Margiotta, Annarita; Martinez-Mora, Juan Antonio; Meli, Athina; Montaruli, Teresa; Moscoso, Luciano; Motz, Holger; Neff, Max; Nezri, Emma Nuel; Palioselitis, Dimitris; Păvălaş, Gabriela E; Payet, Kevin; Payre, Patrice; Petrovic, Jelena; Piattelli, Paolo; Picot-Clemente, Nicolas; Popa, Vlad; Pradier, Thierry; Presani, Eleonora; Racca, Chantal; Reed, Corey; Riccobene, Giorgio; Richardt, Carsten; Richter, Roland; Rivière, Colas; Roensch, Kathrin; Rostovtsev, Andrei; Ruiz-Rivas, Joaquin; Rujoiu, Marius; Russo, Valerio G; Salesa, Francisco; Sánchez-Losa, Augustin; Sapienza, Piera; Schöck, Friederike; Schuller, Jean-Pierre; Schussler, Fabian; Shanidze, Rezo; Simeone, Francesco; Spies, Andreas; Spurio, Maurizio; Steijger, Jos J M; Stolarczyk, Thierry; Taiuti, Mauro G F; Toscano, Simona; Vallage, Bertrand; Van Elewyck, Véronique; Vannoni, Giulia; Vecchi, Manuela; Vernin, Pascal; Wijnker, Guus; Wilms, Jorn; de Wolf, Els; Yepes, Harold; Zaborov, Dmitry; De Dios Zornoza, Juan; Zúñiga, Juan

2013-01-01

The deep ocean is the largest and least known ecosystem on Earth. It hosts numerous pelagic organisms, most of which are able to emit light. Here we present a unique data set consisting of a 2.5-year long record of light emission by deep-sea pelagic organisms, measured from December 2007 to June 2010 at the ANTARES underwater neutrino telescope in the deep NW Mediterranean Sea, jointly with synchronous hydrological records. This is the longest continuous time-series of deep-sea bioluminescence ever recorded. Our record reveals several weeks long, seasonal bioluminescence blooms with light intensity up to two orders of magnitude higher than background values, which correlate to changes in the properties of deep waters. Such changes are triggered by the winter cooling and evaporation experienced by the upper ocean layer in the Gulf of Lion that leads to the formation and subsequent sinking of dense water through a process known as "open-sea convection". It episodically renews the deep water of the study area and conveys fresh organic matter that fuels the deep ecosystems. Luminous bacteria most likely are the main contributors to the observed deep-sea bioluminescence blooms. Our observations demonstrate a consistent and rapid connection between deep open-sea convection and bathypelagic biological activity, as expressed by bioluminescence. In a setting where dense water formation events are likely to decline under global warming scenarios enhancing ocean stratification, in situ observatories become essential as environmental sentinels for the monitoring and understanding of deep-sea ecosystem shifts.

Deep-Sea Bioluminescence Blooms after Dense Water Formation at the Ocean Surface

PubMed Central

Tamburini, Christian; Canals, Miquel; Durrieu de Madron, Xavier; Houpert, Loïc; Lefèvre, Dominique; Martini, Séverine; D'Ortenzio, Fabrizio; Robert, Anne; Testor, Pierre; Aguilar, Juan Antonio; Samarai, Imen Al; Albert, Arnaud; André, Michel; Anghinolfi, Marco; Anton, Gisela; Anvar, Shebli; Ardid, Miguel; Jesus, Ana Carolina Assis; Astraatmadja, Tri L.; Aubert, Jean-Jacques; Baret, Bruny; Basa, Stéphane; Bertin, Vincent; Biagi, Simone; Bigi, Armando; Bigongiari, Ciro; Bogazzi, Claudio; Bou-Cabo, Manuel; Bouhou, Boutayeb; Bouwhuis, Mieke C.; Brunner, Jurgen; Busto, José; Camarena, Francisco; Capone, Antonio; Cârloganu, Christina; Carminati, Giada; Carr, John; Cecchini, Stefano; Charif, Ziad; Charvis, Philippe; Chiarusi, Tommaso; Circella, Marco; Coniglione, Rosa; Costantini, Heide; Coyle, Paschal; Curtil, Christian; Decowski, Patrick; Dekeyser, Ivan; Deschamps, Anne; Donzaud, Corinne; Dornic, Damien; Dorosti, Hasankiadeh Q.; Drouhin, Doriane; Eberl, Thomas; Emanuele, Umberto; Ernenwein, Jean-Pierre; Escoffier, Stéphanie; Fermani, Paolo; Ferri, Marcelino; Flaminio, Vincenzo; Folger, Florian; Fritsch, Ulf; Fuda, Jean-Luc; Galatà, Salvatore; Gay, Pascal; Giacomelli, Giorgio; Giordano, Valentina; Gómez-González, Juan-Pablo; Graf, Kay; Guillard, Goulven; Halladjian, Garadeb; Hallewell, Gregory; van Haren, Hans; Hartman, Joris; Heijboer, Aart J.; Hello, Yann; Hernández-Rey, Juan Jose; Herold, Bjoern; Hößl, Jurgen; Hsu, Ching-Cheng; de Jong, Marteen; Kadler, Matthias; Kalekin, Oleg; Kappes, Alexander; Katz, Uli; Kavatsyuk, Oksana; Kooijman, Paul; Kopper, Claudio; Kouchner, Antoine; Kreykenbohm, Ingo; Kulikovskiy, Vladimir; Lahmann, Robert; Lamare, Patrick; Larosa, Giuseppina; Lattuada, Dario; Lim, Gordon; Presti, Domenico Lo; Loehner, Herbert; Loucatos, Sotiris; Mangano, Salvatore; Marcelin, Michel; Margiotta, Annarita; Martinez-Mora, Juan Antonio; Meli, Athina; Montaruli, Teresa; Motz, Holger; Neff, Max; Nezri, Emma nuel; Palioselitis, Dimitris; Păvălaş, Gabriela E.; Payet, Kevin; Payre, Patrice; Petrovic, Jelena; Piattelli, Paolo; Picot-Clemente, Nicolas; Popa, Vlad; Pradier, Thierry; Presani, Eleonora; Racca, Chantal; Reed, Corey; Riccobene, Giorgio; Richardt, Carsten; Richter, Roland; Rivière, Colas; Roensch, Kathrin; Rostovtsev, Andrei; Ruiz-Rivas, Joaquin; Rujoiu, Marius; Russo, Valerio G.; Salesa, Francisco; Sánchez-Losa, Augustin; Sapienza, Piera; Schöck, Friederike; Schuller, Jean-Pierre; Schussler, Fabian; Shanidze, Rezo; Simeone, Francesco; Spies, Andreas; Spurio, Maurizio; Steijger, Jos J. M.; Stolarczyk, Thierry; Taiuti, Mauro G. F.; Toscano, Simona; Vallage, Bertrand; Van Elewyck, Véronique; Vannoni, Giulia; Vecchi, Manuela; Vernin, Pascal; Wijnker, Guus; Wilms, Jorn; de Wolf, Els; Yepes, Harold; Zaborov, Dmitry; De Dios Zornoza, Juan; Zúñiga, Juan

2013-01-01

The deep ocean is the largest and least known ecosystem on Earth. It hosts numerous pelagic organisms, most of which are able to emit light. Here we present a unique data set consisting of a 2.5-year long record of light emission by deep-sea pelagic organisms, measured from December 2007 to June 2010 at the ANTARES underwater neutrino telescope in the deep NW Mediterranean Sea, jointly with synchronous hydrological records. This is the longest continuous time-series of deep-sea bioluminescence ever recorded. Our record reveals several weeks long, seasonal bioluminescence blooms with light intensity up to two orders of magnitude higher than background values, which correlate to changes in the properties of deep waters. Such changes are triggered by the winter cooling and evaporation experienced by the upper ocean layer in the Gulf of Lion that leads to the formation and subsequent sinking of dense water through a process known as “open-sea convection”. It episodically renews the deep water of the study area and conveys fresh organic matter that fuels the deep ecosystems. Luminous bacteria most likely are the main contributors to the observed deep-sea bioluminescence blooms. Our observations demonstrate a consistent and rapid connection between deep open-sea convection and bathypelagic biological activity, as expressed by bioluminescence. In a setting where dense water formation events are likely to decline under global warming scenarios enhancing ocean stratification, in situ observatories become essential as environmental sentinels for the monitoring and understanding of deep-sea ecosystem shifts. PMID:23874425

[Establishment and application of a Vero cell line stably expressing raccoon dog SLAM, the cellular receptor of canine distemper virus].

PubMed

Zhao, Jianjun; Yan, Ruxun; Zhang, Hailing; Zhang, Lei; Hu, Bo; Bai, Xue; Shao, Xiqun; Chai, Xiuli; Yan, Xijun; Wu, Wei

2012-12-04

The signaling lymphocyte activation molecule (SLAM, also known as CD150), is used as a cellular receptor by canine distemper virus (CDV). Wild-type strains of CDVs can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. Our aim is to establish a Vero cells expressing raccoon dog SLAM (rSLAM) to efficiently isolate CDV from pathological samples. A eukaryotic expression plasmid, pIRES2-EGFP-rSLAMhis, containing rSLAM gene fused with six histidine-coding sequence, EGFP gene, and neomycin resistance gene was constructed. After transfection with the plasmid, a stable cell line, Vero-rSLAM, was screened from Vero cells with the identification of EGFP reporter and G418 resistance. Three CD positive specimens from infected foxes and raccoon dogs were inoculated to Vero-rSLAM cells for CDV isolation. Foxes and raccoon dogs were inoculated subcutaneously LN (10)fl strain with 4 x 10(2.39)TCID50 dose to evaluate pathogenicity of CDV isolations. The rSLAMh fused gene was shown to transcript and express stably in Vero-rSLAM cells by RT-PCR and Immunohistochemistry assay. Three CDV strains were isolated successfully in Vero-rSLAM cells 36 -48 hours after inoculation with spleen or lung specimens from foxes and raccoon dogs with distemper. By contrast, no CDV was recovered from those CD positive specimens when Vero cells were used for virus isolation. Infected foxes and raccoon dogs with LN(10)f1 strain all showed typical CD symptoms and high mortality (2/3 for foxes and 3/3 for raccoon dogs) in 22 days post challenge. Our results indicate that Vero-rSLAM cells stably expressing raccoon dog SLAM are highly sensitive to CDV in clinical specimens and the CDV isolation can maintain high virulence to its host animals.

Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation.

PubMed

Bugeon, Stéphane; de Chevigny, Antoine; Boutin, Camille; Coré, Nathalie; Wild, Stefan; Bosio, Andreas; Cremer, Harold; Beclin, Christophe

2017-11-01

In vivo brain electroporation of DNA expression vectors is a widely used method for lineage and gene function studies in the developing and postnatal brain. However, transfection efficiency of DNA is limited and adult brain tissue is refractory to electroporation. Here, we present a systematic study of mRNA as a vector for acute genetic manipulation in the developing and adult brain. We demonstrate that mRNA electroporation is far more efficient than DNA electroporation, and leads to faster and more homogeneous protein expression in vivo Importantly, mRNA electroporation allows the manipulation of neural stem cells and postmitotic neurons in the adult brain using minimally invasive procedures. Finally, we show that this approach can be efficiently used for functional studies, as exemplified by transient overexpression of the neurogenic factor Myt1l and by stably inactivating Dicer nuclease in vivo in adult born olfactory bulb interneurons and in fully integrated cortical projection neurons. © 2017. Published by The Company of Biologists Ltd.

Enhancing magnetic nanoparticle-based DNA transfection: Intracellular-active cassette features

NASA Astrophysics Data System (ADS)

Vernon, Matthew Martin

Efficient plasmid DNA transfection of embryonic stem cells, mesenchymal stem cells, neural cell lines and the majority of primary cell lines is a current challenge in gene therapy research. Magnetic nanoparticle-based DNA transfection is a gene vectoring technique that is promising because it is capable of outperforming most other non-viral transfection methods in terms of both transfection efficiency and cell viability. The nature of the DNA vector implemented depends on the target cell phenotype, where the particle surface chemistry and DNA binding/unbinding kinetics of the DNA carrier molecule play a critical role in the many steps required for successful gene transfection. Accordingly, Neuromag, an iron oxide/polymer nanoparticle optimized for transfection of neural phenotypes, outperforms many other nanoparticles and lipidbased DNA carriers. Up to now, improvements to nanomagnetic transfection techniques have focused mostly on particle functionalization and transfection parameter optimization (cell confluence, growth media, serum starvation, magnet oscillation parameters, etc.). None of these parameters are capable of assisting the nuclear translocation of delivered plasmid DNA once the particle-DNA complex is released from the endosome and dissociates in the cell's cytoplasm. In this study, incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid DNA confers improved nuclear translocation, demonstrating significant improvement in nanomagnetic transfection efficiency in differentiated SH-SY5Y neuroblastoma cells. Other parameters, such as days in vitro, are also found to play a role and represent potential targets for further optimization.

Monitoring Bloom Dynamics of a Common Coastal Bioluminescent Ctenophore

DTIC Science & Technology

2007-09-30

jellyfish blooms are not well understood, but are generally assumed to be a combination of physical and biological factors, with temperature and...bioluminescent jellyfish , especially of Mnemiopsis leidyi, are a common occurrence that appear to be on the rise. Evidence indicates that these blooms

Disposable bioluminescence-based biosensor for detection of bacterial count in food.

PubMed

Luo, Jinping; Liu, Xiaohong; Tian, Qing; Yue, Weiwei; Zeng, Jing; Chen, Guangquan; Cai, Xinxia

2009-11-01

A biosensor for rapid detection of bacterial count based on adenosine 5'-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin-luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parameters affecting the amount of the extracted ATP, including the types of ATP extractants, the concentrations of ATP extractant, and the relevant neutralizing reagent, were optimized. Under the optimal experimental conditions, the biosensor showed a linear response to standard bacteria in a concentration range from 10(3) to 10(8) colony-forming units (CFU) per milliliter with a correlation coefficient of 0.925 (n=22) within 5min. Moreover, the bacterial count of real food samples obtained by the biosensor correlated well with those by the conventional plate count method. The proposed biosensor, with characteristics of low cost, easy operation, and fast response, provides potential application to rapid evaluation of bacterial contamination in the food industry, environment monitoring, and other fields.

Smartphone-based low light detection for bioluminescence application.

PubMed

Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

2017-01-09

We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~10 6  CFU/mL of the bio-reporter, which corresponds to ~10 7 photons/s with 180 seconds of integration time.

Smartphone-based low light detection for bioluminescence application

NASA Astrophysics Data System (ADS)

Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

2017-01-01

We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~106 CFU/mL of the bio-reporter, which corresponds to ~107 photons/s with 180 seconds of integration time.

Smartphone-based low light detection for bioluminescence application

PubMed Central

Kim, Huisung; Jung, Youngkee; Doh, Iyll-Joon; Lozano-Mahecha, Roxana Andrea; Applegate, Bruce; Bae, Euiwon

2017-01-01

We report a smartphone-based device and associated imaging-processing algorithm to maximize the sensitivity of standard smartphone cameras, that can detect the presence of single-digit pW of radiant flux intensity. The proposed hardware and software, called bioluminescent-based analyte quantitation by smartphone (BAQS), provides an opportunity for onsite analysis and quantitation of luminescent signals from biological and non-biological sensing elements which emit photons in response to an analyte. A simple cradle that houses the smartphone, sample tube, and collection lens supports the measuring platform, while noise reduction by ensemble averaging simultaneously lowers the background and enhances the signal from emitted photons. Five different types of smartphones, both Android and iOS devices, were tested, and the top two candidates were used to evaluate luminescence from the bioluminescent reporter Pseudomonas fluorescens M3A. The best results were achieved by OnePlus One (android), which was able to detect luminescence from ~106 CFU/mL of the bio-reporter, which corresponds to ~107 photons/s with 180 seconds of integration time. PMID:28067287

Optical biosensor for environmental on-line monitoring of naphthalene and salicylate bioavailability with an immobilized bioluminescent catabolic reporter bacterium.

PubMed Central

Heitzer, A; Malachowsky, K; Thonnard, J E; Bienkowski, P R; White, D C; Sayler, G S

1994-01-01

An optical whole-cell biosensor based on a genetically engineered bioluminescent catabolic reporter bacterium was developed for continuous on-line monitoring of naphthalene and salicylate bioavailability and microbial catabolic activity potential in waste streams. The bioluminescent reporter bacterium, Pseudomonas fluorescens HK44, carries a transcriptional nahG-luxCDABE fusion for naphthalene and salicylate catabolism. Exposure to either compound resulted in inducible bioluminescence. The reporter culture was immobilized onto the surface of an optical light guide by using strontium alginate. This biosensor probe was then inserted into a measurement cell which simultaneously received the waste stream solution and a maintenance medium. Exposure under defined conditions to both naphthalene and salicylate resulted in a rapid increase in bioluminescence. The magnitude of the response and the response time were concentration dependent. Good reproducibility of the response was observed during repetitive perturbations with either naphthalene or salicylate. Exposure to other compounds, such as glucose and complex nutrient medium or toluene, resulted in either minor bioluminescence increases after significantly longer response times compared with naphthalene or no response, respectively. The environmental utility of the biosensor was tested by using real pollutant mixtures. A specific bioluminescence response was obtained after exposure to either an aqueous solution saturated with JP-4 jet fuel or an aqueous leachate from a manufactured-gas plant soil, since naphthalene was present in both pollutant mixtures. PMID:8017932

Turbulent circulation above the surface heat source in stably stratified atmosphere

NASA Astrophysics Data System (ADS)

Kurbatskii, A. F.; Kurbatskaya, L. I.

2016-10-01

The 3-level RANS approach for simulating a turbulent circulation over the heat island in a stably stratified environment under nearly calm conditions is formulated. The turbulent kinetic energy its spectral consumption (dissipation) and the dispersion of turbulent fluctuations of temperature are found from differential equations, thus the correct modeling of transport processes in the interface layer with the counter-gradient heat flux is assured. The three-parameter turbulence RANS approach minimizes difficulties in simulating the turbulent transport in a stably stratified environment and reduces efforts needed for the numerical implementation of the 3-level RANS approach. Numerical simulation of the turbulent structure of the penetrative convection over the heat island under conditions of stably stratified atmosphere demonstrates that the three-equation model is able to predict the thermal circulation induced by the heat island. The temperature distribution, root-mean-square fluctuations of the turbulent velocity and temperature fields and spectral turbulent kinetic energy flux are in good agreement with the experimental data. The model describes such thin physical effects, as a crossing of vertical profiles of temperature of a thermal plume with the formation of the negative buoyancy area testifying to development of the dome-shaped form at the top part of a plume in the form of "hat".

Comparison of chemotherapeutic drug resistance in cells transfected with canine ABCG2 or feline ABCG2.

PubMed

Lewis, R S; Fidel, J; Dassanayake, S; Court, M H; Burke, N S; Mealey, K L

2017-06-01

ABCG2 (ATP binding cassette subfamily G, member 2) mediates resistance to a variety of cytotoxic agents. Although human ABCG2 is well characterized, the function of canine ABCG2 has not been studied previously. Feline ABCG2 has an amino acid substitution in the adenosine triphosphate-binding domain that decreases its transport capacity relative to human ABCG2. Our goal was to compare canine ABCG2-mediated chemotherapeutic drug resistance to feline ABCG2-mediated chemotherapeutic drug resistance. HEK-293 cells stably transfected with plasmid containing canine ABCG2, feline ABCG2 or no ABCG2 were exposed to carboplatin, doxorubicin, mitoxantrone, toceranib or vincristine, and cell survival was subsequently determined. Canine ABCG2 conferred a greater degree of chemotherapy resistance than feline ABCG2 for mitoxantrone. Neither canine nor feline ABCG2 conferred resistance to doxorubicin, vincristine or toceranib. Canine, but not feline, ABCG2 conferred resistance to carboplatin, a drug that is not reported to be a substrate for ABCG2 in other species. © 2015 John Wiley & Sons Ltd.

Melatonin Entrains PER2::LUC Bioluminescence Circadian Rhythm in the Mouse Cornea

PubMed Central

Baba, Kenkichi; Davidson, Alec J.; Tosini, Gianluca

2015-01-01

Purpose Previous studies have reported the presence of a circadian rhythm in PERIOD2::LUCIFERASE (PER2::LUC) bioluminescence in mouse photoreceptors, retina, RPE, and cornea. Melatonin (MLT) modulates many physiological functions in the eye and it is believed to be one of the key circadian signals within the eye. The aim of the present study was to investigate the regulation of the PER2::LUC circadian rhythm in mouse cornea and to determine the role played by MLT. Methods Corneas were obtained from PER2::LUC mice and cultured to measure bioluminescence rhythmicity in isolated tissue using a Lumicycle or CCD camera. To determine the time-dependent resetting of the corneal circadian clocks in response to MLT or IIK7 (a melatonin type 2 receptor, MT2, agonist) was added to the cultured corneas at different times of the day. We also defined the location of the MT2 receptor within different corneal layers using immunohistochemistry. Results A long-lasting bioluminescence rhythm was recorded from cultured PER2::LUC cornea and PER2::LUC signal was localized to the corneal epithelium and endothelium. MLT administration in the early night delayed the cornea rhythm, whereas administration of MLT at late night to early morning advanced the cornea rhythm. Treatment with IIK7 mimicked the MLT phase-shifting effect. Consistent with these results, MT2 immunoreactivity was localized to the corneal epithelium and endothelium. Conclusions Our work demonstrates that MLT entrains the PER2::LUC bioluminescence rhythm in the cornea. Our data indicate that the cornea may represent a model to study the molecular mechanisms by which MLT affects the circadian clock. PMID:26207312

Bioluminescence Truth Data Measurement and Signature Detection

DTIC Science & Technology

2006-01-01

bioluminescence activity and related forcing factors. Kilroy sensors are shown attached to pilings with the senor system below water and the cell phone based...communications module attached to the top of the piling. A cell phone tower represents communication of data to shore. Also shown are distributed...installation are located based on GPS coordinates telemetered by the cell phone module. Icons point in direction of most recently measured flow and

Theoretical tuning of the firefly bioluminescence spectra by the modification of oxyluciferin

NASA Astrophysics Data System (ADS)

Cheng, Yuan-Yuan; Zhu, Jia; Liu, Ya-Jun

2014-01-01

Extending the firefly bioluminescence is of practical significance for the improved visualization of living cells and the development of a multicolor reporter. Tuning the color of bioluminescence in fireflies mainly involves the modification of luciferase and luciferin. In this Letter, we theoretically studied the emission spectra of 9 firefly oxyluciferin analogs in the gas phase and in solutions. Three density functionals, including B3LYP, CAM-B3LYP and M06-2X, were employed to theoretically predict the efficiently luminescent analogs. The reliable functionals for calculating the targeted systems were suggested. The luminescence efficiency, solvent effects, and substituent effects are discussed based on the calculated results.

Sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase.

PubMed

Mezzanotte, Laura; Que, Ivo; Kaijzel, Eric; Branchini, Bruce; Roda, Aldo; Löwik, Clemens

2011-04-22

Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time.

Sensitive Dual Color In Vivo Bioluminescence Imaging Using a New Red Codon Optimized Firefly Luciferase and a Green Click Beetle Luciferase

PubMed Central

Mezzanotte, Laura; Que, Ivo; Kaijzel, Eric; Branchini, Bruce; Roda, Aldo; Löwik, Clemens

2011-01-01

Background Despite a plethora of bioluminescent reporter genes being cloned and used for cell assays and molecular imaging purposes, the simultaneous monitoring of multiple events in small animals is still challenging. This is partly attributable to the lack of optimization of cell reporter gene expression as well as too much spectral overlap of the color-coupled reporter genes. A new red emitting codon-optimized luciferase reporter gene mutant of Photinus pyralis, Ppy RE8, has been developed and used in combination with the green click beetle luciferase, CBG99. Principal Findings Human embryonic kidney cells (HEK293) were transfected with vectors that expressed red Ppy RE8 and green CBG99 luciferases. Populations of red and green emitting cells were mixed in different ratios. After addition of the shared single substrate, D-luciferin, bioluminescent (BL) signals were imaged with an ultrasensitive cooled CCD camera using a series of band pass filters (20 nm). Spectral unmixing algorithms were applied to the images where good separation of signals was observed. Furthermore, HEK293 cells that expressed the two luciferases were injected at different depth in the animals. Spectrally-separate images and quantification of the dual BL signals in a mixed population of cells was achieved when cells were either injected subcutaneously or directly into the prostate. Significance We report here the re-engineering of different luciferase genes for in vitro and in vivo dual color imaging applications to address the technical issues of using dual luciferases for imaging. In respect to previously used dual assays, our study demonstrated enhanced sensitivity combined with spatially separate BL spectral emissions using a suitable spectral unmixing algorithm. This new D-luciferin-dependent reporter gene couplet opens up the possibility in the future for more accurate quantitative gene expression studies in vivo by simultaneously monitoring two events in real time. PMID:21544210

pEGFP transfection into murine skeletal muscle by electrosonoporation

NASA Astrophysics Data System (ADS)

Tamošiūnas, Mindaugas; Jakovels, Dainis; Rubins, Uldis; Kadikis, Roberts; Petrovska, Ramona; Šatkauskas, Saulius

2017-12-01

In this study, we aimed to determine whether the combination of electroporation (EP) and ultrasound (US) waves (sonoporation) can affect the plasmid DNA transfection to mice tibialis cranialis muscle. Multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) fluorescence, providing information on location and duration of EGFP expression. We found that electrosonoporation, commonly enhancing pDNA transfection in vitro, had no positive effect on EGFP transfection efficiency increase in vivo with respect to electroporation alone. We presume that this may be associated with decreased viability of transfected fibers.

Development of Bioluminescent Cronobacter sakazakii ATCC 29544 in a Mouse Model.

PubMed

Wang, Xiwen; Li, Zhiping; Dong, Xiaolin; Chi, Hang; Wang, Guannan; Li, Jiakuan; Sun, Rui; Chen, Man; Zhang, Xinying; Wang, Yuanyuan; Qu, Han; Sun, Yu; Xia, Zhiping; Li, Qianxue

2015-05-01

Cronobacter sakazakii is an emerging pathogen that causes severe and life-threatening conditions including meningitis, bacteremia, and necrotizing enterocolitis. An animal model study for extrapolation of C. sakazakii infection can provide a better understanding of pathogenesis. However, methods for real-time monitoring of the course of C. sakazakii infection in living animals have been lacking. We developed a bioluminescent C. sakazakii strain (ATCC 29544) that can be used for real-time monitoring of C. sakazakii infection in BALB/c mice. C. sakazakii ATCC 29544 mainly colonized brain, liver, spleen, kidney, and gastrointestinal tract, as indicated by bioluminescence imaging. This work provides a novel approach for studying the progression of C. sakazakii infection and evaluating therapeutics in a living mouse model.

Regulated bioluminescence as a tool for bioremediation process monitoring and control of bacterial cultures

NASA Technical Reports Server (NTRS)

Burlage, Robert S.; Heitzer, Armin; Digrazia, Philip M.

1991-01-01

An effective on-line monitoring technique for toxic waste bioremediation using bioluminescent microorganisms has shown great potential for the description and optimization of biological processes. The lux genes of the bacterium Vibrio fischeri are used by this species to produce visible light. The lux genes can be genetically fused to the control region of a catabolic gene, with the result that bioluminescence is produced whenever the catabolic gene is induced. Thus the detection of light from a sample indicates that genetic expression from a specific gene is occurring. This technique was used to monitor biodegradation of specific contaminants from waste sites. For these studies, fusions between the lux genes and the operons for naphthalene and toluene/xylene degradation were constructed. Strains carrying one of these fusions respond sensitively and specifically to target substrates. Bioluminescence from these cultures can be rapidly measured in a nondestructive and noninvasive manner. The potential for this technique in this and other biological systems is discussed.

Bioluminescence ATP Monitoring for the Routine Assessment of Food Contact Surface Cleanliness in a University Canteen

PubMed Central

Osimani, Andrea; Garofalo, Cristiana; Clementi, Francesca; Tavoletti, Stefano; Aquilanti, Lucia

2014-01-01

ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli) were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP) at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs), including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99) between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene. PMID:25329534

Bioluminescence ATP monitoring for the routine assessment of food contact surface cleanliness in a university canteen.

PubMed

Osimani, Andrea; Garofalo, Cristiana; Clementi, Francesca; Tavoletti, Stefano; Aquilanti, Lucia

2014-10-17

ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli) were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP) at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs), including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99) between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene.

Hybrid light transport model based bioluminescence tomography reconstruction for early gastric cancer detection

NASA Astrophysics Data System (ADS)

Chen, Xueli; Liang, Jimin; Hu, Hao; Qu, Xiaochao; Yang, Defu; Chen, Duofang; Zhu, Shouping; Tian, Jie

2012-03-01

Gastric cancer is the second cause of cancer-related death in the world, and it remains difficult to cure because it has been in late-stage once that is found. Early gastric cancer detection becomes an effective approach to decrease the gastric cancer mortality. Bioluminescence tomography (BLT) has been applied to detect early liver cancer and prostate cancer metastasis. However, the gastric cancer commonly originates from the gastric mucosa and grows outwards. The bioluminescent light will pass through a non-scattering region constructed by gastric pouch when it transports in tissues. Thus, the current BLT reconstruction algorithms based on the approximation model of radiative transfer equation are not optimal to handle this problem. To address the gastric cancer specific problem, this paper presents a novel reconstruction algorithm that uses a hybrid light transport model to describe the bioluminescent light propagation in tissues. The radiosity theory integrated with the diffusion equation to form the hybrid light transport model is utilized to describe light propagation in the non-scattering region. After the finite element discretization, the hybrid light transport model is converted into a minimization problem which fuses an l1 norm based regularization term to reveal the sparsity of bioluminescent source distribution. The performance of the reconstruction algorithm is first demonstrated with a digital mouse based simulation with the reconstruction error less than 1mm. An in situ gastric cancer-bearing nude mouse based experiment is then conducted. The primary result reveals the ability of the novel BLT reconstruction algorithm in early gastric cancer detection.

Optical transfection using an endoscope-like system.

PubMed

Ma, Nan; Gunn-Moore, Frank; Dholakia, Kishan

2011-02-01

Optical transfection is a powerful method for targeted delivery of therapeutic agents to biological cells. A tightly focused pulsed laser beam may transiently change the permeability of a cell membrane to facilitate the delivery of foreign genetic material into cells. We report the first realization of an endoscope-like integrated system for optical transfection. An imaging fiber (coherent optical fiber bundle) with ∼ 6000 cores (pixels) embedded in a fiber cladding of ∼ 300 μm in diameter, produces an image circle (area) of ∼ 270 μm diam. This imaging fiber, with an ordered axicon lens array chemically etched at its exit face, is used for the delivery of a femtosecond laser to the cell membrane for optical transfection along with subcellular resolution imaging. A microcapillary-based microfluidic system for localized drug delivery was also combined in this miniature, flexible system. Using this novel system, a plasmid transfection efficiency up to ∼ 72% was obtained for CHO-K1 cells. This endoscope-like system opens a range of exciting applications, in particular, in the targeted in vivo optical microsurgery area.

Screening for (anti)androgenic properties using a standard operation protocol based on the human stably transfected androgen sensitive PALM cell line. First steps towards validation.

PubMed

Freyberger, A; Witters, H; Weimer, M; Lofink, W; Berckmans, P; Ahr, H-J

2010-08-01

Despite more than a decade of research in the field of endocrine active compounds targeting the androgen receptor (AR), and although suitable cell lines can be obtained, no validated human stably transfected androgen sensitive transactivation assay is available. Bayer Schering Pharma (BSP) and the Flemish Institute for Technological Research (VITO), partners within the EU-sponsored 6th framework project ReProTect, made first steps towards such a validation. A standard operation protocol (SOP) developed at BSP based on the androgen sensitive PALM cell line was transferred to VITO and its performance and transferability were thoroughly studied. The investigation followed a generic protocol prepared for all reporter gene assays evaluated within ReProTect, and in both laboratories at least three independent experiments were performed. The highest concentration to be tested was limited to 10 microM, if needed. A few compounds, 17alpha-methyltestosterone (17alpha-MT), vinclozolin and linuron, were studied using a real world scenario, i.e., assuming that their interaction with the AR was not known: A prescreening for agonism and true, competitive antagonism was used to select conditions such as the appropriate mode of action, and the working range excluding cytotoxicity for the final screening. All other compounds were tested according to the generic protocol: Compounds screened for agonism were the reference androgen 17alpha-methyldihydrotestosterone (MDHT), levonorgestrel, norethynodrel, progesterone, o,p'-DDT, and dibutylphthalate (DBP), while compounds screened for antagonism were the reference anti-androgen flutamide, prochloraz, o,p'-DDT, progesterone, norethynodrel, and DBP. Cytotoxicity was assessed in parallel as lactate dehydrogenase release. The prescreen classified 17alpha-MT as androgenic, vinclozolin and linuron as anti-androgenic and compounds were tested accordingly. In the absence of cytotoxicity, appropriate androgenic properties of reference and test

Diversity of the luciferin binding protein gene in bioluminescent dinoflagellates--insights from a new gene in Noctiluca scintillans and sequences from gonyaulacoid genera.

PubMed

Valiadi, Martha; Iglesias-Rodriguez, Maria Debora

2014-01-01

Dinoflagellate bioluminescence systems operate with or without a luciferin binding protein, representing two distinct modes of light production. However, the distribution, diversity, and evolution of the luciferin binding protein gene within bioluminescent dinoflagellates are not well known. We used PCR to detect and partially sequence this gene from the heterotrophic dinoflagellate Noctiluca scintillans and a group of ecologically important gonyaulacoid species. We report an additional luciferin binding protein gene in N. scintillans which is not attached to luciferase, further to its typical combined bioluminescence gene. This supports the hypothesis that a profound re-organization of the bioluminescence system has taken place in this organism. We also show that the luciferin binding protein gene is present in the genera Ceratocorys, Gonyaulax, and Protoceratium, and is prevalent in bioluminescent species of Alexandrium. Therefore, this gene is an integral component of the standard molecular bioluminescence machinery in dinoflagellates. Nucleotide sequences showed high within-strain variation among gene copies, revealing a highly diverse gene family comprising multiple gene types in some organisms. Phylogenetic analyses showed that, in some species, the evolution of the luciferin binding protein gene was different from the organism's general phylogenies, highlighting the complex evolutionary history of dinoflagellate bioluminescence systems. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.

PubMed

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Magnetic cell sorting purification of differentiated embryonic stem cells stably expressing truncated human CD4 as surface marker.

PubMed

David, Robert; Groebner, Michael; Franz, Wolfgang-Michael

2005-04-01

Embryonic stem (ES) cells offer great potential in regenerative medicine and tissue engineering. Clinical applications are still hampered by the lack of protocols for gentle, high-yield isolation of specific cell types for transplantation expressing no immunogenic markers. We describe labeling of stably transfected ES cells expressing a human CD4 molecule lacking its intracellular domain (DeltaCD4) under control of the phosphoglycerate kinase promoter for magnetic cell sorting (MACS). To track the labeled ES cells, we fused DeltaCD4 to an intracellular enhanced green fluorescent protein domain (DeltaCD4EGFP). We showed functionality of the membrane-bound fluorescent fusion protein and its suitability for MACS leading to purities greater than 97%. Likewise, expression of DeltaCD4 yielded up to 98.5% positive cells independently of their differentiation state. Purities were not limited by the initial percentage of DeltaCD4(+) cells, ranging from 0.6%-16%. The viability of MACS-selected cells was demonstrated by reaggregation and de novo formation of embryoid bodies developing all three germ layers. Thus, expression of DeltaCD4 in differentiated ES cells may enable rapid, high-yield purification of a desired cell type for tissue engineering and transplantation studies.

Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

PubMed

Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

2003-07-15

Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

Brominated Luciferins Are Versatile Bioluminescent Probes

DOE PAGES

Steinhardt, Rachel C.; Rathbun, Colin M.; Krull, Brandon T.; ...

2016-12-08

Here, we report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.

Robust red-emission spectra and yields in firefly bioluminescence against temperature changes

NASA Astrophysics Data System (ADS)

Mochizuki, Toshimitsu; Wang, Yu; Hiyama, Miyabi; Akiyama, Hidefumi

2014-05-01

We measured the quantitative spectra of firefly (Photinus pyralis) bioluminescence at various temperatures to investigate the temperature dependence of the luciferin-luciferase reaction at 15-34 °C. The quantitative spectra were decomposed very well into red (1.9 eV), orange (2.0 eV), and green (2.2 eV) Gaussian components. The intensity of the green component was the only temperature sensitive quantity that linearly decreased as the temperature increased at pH 7 and 8. We found the quantitative bioluminescence spectra to be robust below 2.0 eV against temperature and other experimental conditions. The revealed robustness of the red emissions should be useful for quantitative applications such as adenosine-5'-triphosphate detection.

Bioluminescent signals spatially amplified by wavelength-specific diffusion through the shell of a marine snail.

PubMed

Deheyn, Dimitri D; Wilson, Nerida G

2011-07-22

Some living organisms produce visible light (bioluminescence) for intra- or interspecific visual communication. Here, we describe a remarkable bioluminescent adaptation in the marine snail Hinea brasiliana. This species produces a luminous display in response to mechanical stimulation caused by encounters with other motile organisms. The light is produced from discrete areas on the snail's body beneath the snail's shell, and must thus overcome this structural barrier to be viewed by an external receiver. The diffusion and transmission efficiency of the shell is greater than a commercial diffuser reference material. Most strikingly, the shell, although opaque and pigmented, selectively diffuses the blue-green wavelength of the species bioluminescence. This diffusion generates a luminous display that is enlarged relative to the original light source. This unusual shell thus allows spatially amplified outward transmission of light communication signals from the snail, while allowing the animal to remain safely inside its hard protective shell.

Bioluminescence imaging: a shining future for cardiac regeneration

PubMed Central

Roura, Santiago; Gálvez-Montón, Carolina; Bayes-Genis, Antoni

2013-01-01

Advances in bioanalytical techniques have become crucial for both basic research and medical practice. One example, bioluminescence imaging (BLI), is based on the application of natural reactants with light-emitting capabilities (photoproteins and luciferases) isolated from a widespread group of organisms. The main challenges in cardiac regeneration remain unresolved, but a vast number of studies have harnessed BLI with the discovery of aequorin and green fluorescent proteins. First described in the luminous hydromedusan Aequorea victoria in the early 1960s, bioluminescent proteins have greatly contributed to the design and initiation of ongoing cell-based clinical trials on cardiovascular diseases. In conjunction with advances in reporter gene technology, BLI provides valuable information about the location and functional status of regenerative cells implanted into numerous animal models of disease. The purpose of this review was to present the great potential of BLI, among other existing imaging modalities, to refine effectiveness and underlying mechanisms of cardiac cell therapy. We recount the first discovery of natural primary compounds with light-emitting capabilities, and follow their applications to bioanalysis. We also illustrate insights and perspectives on BLI to illuminate current efforts in cardiac regeneration, where the future is bright. PMID:23402217

Novel mechanism of gene transfection by low-energy shock wave.

PubMed

Ha, Chang Hoon; Lee, Seok Cheol; Kim, Sunghyen; Chung, Jihwa; Bae, Hasuk; Kwon, Kihwan

2015-08-05

Extracorporeal shock wave (SW) therapy has been studied in the transfection of naked nucleic acids into various cell lines through the process of sonoporation, a process that affects the permeation of cell membranes, which can be an effect of cavitation. In this study, siRNAs were efficiently transfected into primary cultured cells and mouse tumor tissue via SW treatment. Furthermore SW-induced siRNA transfection was not mediated by SW-induced sonoporation, but by microparticles (MPs) secreted from the cells. Interestingly, the transfection effect of the siRNAs was transferable through the secreted MPs from human umbilical vein endothelial cell (HUVEC) culture medium after treatment with SW, into HUVECs in another culture plate without SW treatment. In this study, we suggest for the first time a mechanism of gene transfection induced by low-energy SW through secreted MPs, and show that it is an efficient physical gene transfection method in vitro and represents a safe therapeutic strategy for site-specific gene delivery in vivo.

A Multi-Camera System for Bioluminescence Tomography in Preclinical Oncology Research

PubMed Central

Lewis, Matthew A.; Richer, Edmond; Slavine, Nikolai V.; Kodibagkar, Vikram D.; Soesbe, Todd C.; Antich, Peter P.; Mason, Ralph P.

2013-01-01

Bioluminescent imaging (BLI) of cells expressing luciferase is a valuable noninvasive technique for investigating molecular events and tumor dynamics in the living animal. Current usage is often limited to planar imaging, but tomographic imaging can enhance the usefulness of this technique in quantitative biomedical studies by allowing accurate determination of tumor size and attribution of the emitted light to a specific organ or tissue. Bioluminescence tomography based on a single camera with source rotation or mirrors to provide additional views has previously been reported. We report here in vivo studies using a novel approach with multiple rotating cameras that, when combined with image reconstruction software, provides the desired representation of point source metastases and other small lesions. Comparison with MRI validated the ability to detect lung tumor colonization in mouse lung. PMID:26824926

Registration of planar bioluminescence to magnetic resonance and x-ray computed tomography images as a platform for the development of bioluminescence tomography reconstruction algorithms.

PubMed

Beattie, Bradley J; Klose, Alexander D; Le, Carl H; Longo, Valerie A; Dobrenkov, Konstantine; Vider, Jelena; Koutcher, Jason A; Blasberg, Ronald G

2009-01-01

The procedures we propose make possible the mapping of two-dimensional (2-D) bioluminescence image (BLI) data onto a skin surface derived from a three-dimensional (3-D) anatomical modality [magnetic resonance (MR) or computed tomography (CT)] dataset. This mapping allows anatomical information to be incorporated into bioluminescence tomography (BLT) reconstruction procedures and, when applied using sources visible to both optical and anatomical modalities, can be used to evaluate the accuracy of those reconstructions. Our procedures, based on immobilization of the animal and a priori determined fixed projective transforms, should be more robust and accurate than previously described efforts, which rely on a poorly constrained retrospectively determined warping of the 3-D anatomical information. Experiments conducted to measure the accuracy of the proposed registration procedure found it to have a mean error of 0.36+/-0.23 mm. Additional experiments highlight some of the confounds that are often overlooked in the BLT reconstruction process, and for two of these confounds, simple corrections are proposed.

Site-directed mutagenesis of firefly luciferase: implication of conserved residue(s) in bioluminescence emission spectra among firefly luciferases.

PubMed

Tafreshi, Narges Kh; Sadeghizadeh, Majid; Emamzadeh, Rahman; Ranjbar, Bijan; Naderi-Manesh, Hossein; Hosseinkhani, Saman

2008-05-15

The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than green light and being less absorbed by biological tissues, red-emitting luciferases have been considered as useful reporters in imaging technology. A set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase has been made by site-directed mutagenesis. Among different beetle luciferases, those from Phrixothrix (railroad worm) emit either green or red bioluminescence colours naturally. By substitution of three specific amino acids using site-specific mutagenesis in a green-emitting luciferase (from L. turkestanicus), the colour of emitted light was changed to red concomitant with decreasing decay rate. Different specific mutations (H245N, S284T and H431Y) led to changes in the bioluminescence colour. Meanwhile, the luciferase reaction took place with relative retention of its basic kinetic properties such as K(m) and relative activity. Structural comparison of the native and mutant luciferases using intrinsic fluorescence, far-UV CD spectra and homology modelling revealed a significant conformational change in mutant forms. A change in the colour of emitted light indicates the critical role of these conserved residues in bioluminescence colour determination among firefly luciferases. Relatively high specific activity and emission of red light might make these mutants suitable as reporters for the study of gene expression and bioluminescence imaging.

Hydrodynamics based transfection in normal and fibrotic rats

PubMed Central

Yeikilis, Rita; Gal, Shunit; Kopeiko, Natalia; Paizi, Melia; Pines, Mark; Braet, Filip; Spira, Gadi

2006-01-01

AIM: Hydrodynamics based transfection (HBT), the injection of a large volume of naked plasmid DNA in a short time is a relatively simple, efficient and safe method for in vivo transfection of liver cells. Though used for quite some time, the mechanism of gene transfection has not yet been elucidated. METHODS: A luciferase encoding plasmid was injected using the hydrodynamics based procedure into normal and thioacetamide-induced fibrotic Sprague Dawley rats. Scanning and transmission electron microscopy images were taken. The consequence of a dual injection of Ringer solution and luciferase pDNA was followed. Halofuginone, an anti collagen type I inhibitor was used to reduce ECM load in fibrotic rats prior to the hydrodynamic injection. RESULTS: Large endothelial gaps formed as soon as 10’ following hydrodynamic injection; these gradually returned to normal 10 d post injection. Hydrodynamic administration of Ringer 10 or 30 m prior to moderate injection of plasmid did not result in efficient transfection suggesting that endothelial gaps by themselves are not sufficient for gene expression. Gene transfection following hydrodynamic injection in thioacetamide induced fibrotic rats was diminished coinciding with the level of fibrosis. Halofuginone, a specific collagen typeIinhibitor, alleviated this effect. CONCLUSION: The hydrodynamic pressure formed following HBT results in the formation of large endothelial gaps. These gaps, though important in the transfer of DNA molecules from the blood to the space of Disse are not enough to provide the appropriate conditions for hepatocyte transfection. Hydrodynamics based injection is applicable in fibrotic rats provided that ECM load is reduced. PMID:17036386

Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta.

PubMed

Ma, Z; Ramanadham, S; Wohltmann, M; Bohrer, A; Hsu, F F; Turk, J

2001-04-20

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.

Evaluation of different photosensitizers for use in photochemical gene transfection.

PubMed

Prasmickaite, L; Høgset, A; Berg, K

2001-04-01

Many potentially therapeutic macromolecules, e.g. transgenes used in gene therapy, are taken into the cells by endocytosis, and have to be liberated from endocytic vesicles in order to express a therapeutic function. To achieve this we have developed a new technology, named photochemical internalization (PCI), based on photochemical reactions inducing rupture of endocytic vesicles. The aim of this study was to clarify which properties of photosensitizers are important for obtaining the PCI effect improving gene transfection. The photochemical effect on transfection of human melanoma THX cells has been studied employing photosensitizers with different physicochemical properties and using two gene delivery vectors: the cationic polypeptide polylysine and the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Photochemical treatment by photosensitizers that do not localize in endocytic vesicles (tetra[3-hydroxyphenyl]porphyrin and 5-aminolevulinic acid-induced protoporphyrin IX) do not stimulate transfection, irrespective of the gene delivery vector. In contrast, photosensitizers localized in endocytic vesicles stimulate polylysine-mediated transfection, and amphiphilic photosensitizers (disulfonated aluminium phthalocyanine [AlPcS2a] and meso-tetraphenylporphynes) show the strongest positive effect, inducing approximately 10-fold increase in transfection efficiency. In contrast, DOTAP-mediated transfection is inhibited by all photochemical treatments irrespective of the photosensitizer used. Neither AlPcS2a nor Photofrin affects the uptake of the transfecting DNA over the plasma membrane, therefore photochemical permeabilization of endocytic vesicles seems to be the most likely mechanism responsible for the positive PCI effect on gene transfection.

Bioluminescence Tomography–Guided Radiation Therapy for Preclinical Research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Bin; Wang, Ken Kang-Hsin, E-mail: kwang27@jhmi.edu; Yu, Jingjing

Purpose: In preclinical radiation research, it is challenging to localize soft tissue targets based on cone beam computed tomography (CBCT) guidance. As a more effective method to localize soft tissue targets, we developed an online bioluminescence tomography (BLT) system for small-animal radiation research platform (SARRP). We demonstrated BLT-guided radiation therapy and validated targeting accuracy based on a newly developed reconstruction algorithm. Methods and Materials: The BLT system was designed to dock with the SARRP for image acquisition and to be detached before radiation delivery. A 3-mirror system was devised to reflect the bioluminescence emitted from the subject to a stationarymore » charge-coupled device (CCD) camera. Multispectral BLT and the incomplete variables truncated conjugate gradient method with a permissible region shrinking strategy were used as the optimization scheme to reconstruct bioluminescent source distributions. To validate BLT targeting accuracy, a small cylindrical light source with high CBCT contrast was placed in a phantom and also in the abdomen of a mouse carcass. The center of mass (CoM) of the source was recovered from BLT and used to guide radiation delivery. The accuracy of the BLT-guided targeting was validated with films and compared with the CBCT-guided delivery. In vivo experiments were conducted to demonstrate BLT localization capability for various source geometries. Results: Online BLT was able to recover the CoM of the embedded light source with an average accuracy of 1 mm compared to that with CBCT localization. Differences between BLT- and CBCT-guided irradiation shown on the films were consistent with the source localization revealed in the BLT and CBCT images. In vivo results demonstrated that our BLT system could potentially be applied for multiple targets and tumors. Conclusions: The online BLT/CBCT/SARRP system provides an effective solution for soft tissue targeting, particularly for small

Cationic lipids: molecular structure/ transfection activity relationships and interactions with biomembranes.

PubMed

Koynova, Rumiana; Tenchov, Boris

2010-01-01

Abstract Synthetic cationic lipids, which form complexes (lipoplexes) with polyanionic DNA, are presently the most widely used constituents of nonviral gene carriers. A large number of cationic amphiphiles have been synthesized and tested in transfection studies. However, due to the complexity of the transfection pathway, no general schemes have emerged for correlating the cationic lipid chemistry with their transfection efficacy and the approaches for optimizing their molecular structures are still largely empirical. Here we summarize data on the relationships between transfection activity and cationic lipid molecular structure and demonstrate that the transfection activity depends in a systematic way on the lipid hydrocarbon chain structure. A number of examples, including a large series of cationic phosphatidylcholine derivatives, show that optimum transfection is displayed by lipids with chain length of approximately 14 carbon atoms and that the transfection efficiency strongly increases with increase of chain unsaturation, specifically upon replacement of saturated with monounsaturated chains.

Evaluation of the anti-neoplastic effect of sorafenib on liver cancer through bioluminescence tomography

NASA Astrophysics Data System (ADS)

Liang, Qian; Ye, Jinzuo; Du, Yang; Chi, Chongwei; Tian, Jie

2017-03-01

Hepatocellular carcinoma (HCC) is one of the most important leading causes of cancer-related deaths worldwide. In this study, we evaluated the efficacy of sorafenib on hepatocellular carcinoma through bioluminescence tomography (BLT) based on Micro-CT/BLT multi-modal system. Initially, the human hepatocellular carcinoma cell line HepG2-Red-FLuc, which was transfected with luciferase gene, was cultured. And then, the orthotopic liver tumor mouse model was established on 4 5 weeks old athymic male Balb/c nude mice by inoculating the HepG2-Red-FLuc cell suspension into the liver lobe under isoflurane anesthesia. 15 20 days after tumor cells implantation, the mice were divided into two groups including the sorafenib treatment group and the control group. The mice in the treatment group were treated with sorafenib with dosage of 62 mg/kg/day by oral gavage for continuous 14 days, and the mice in the control group were treated with sterile water at equal volume. The tumor growth and drug treatment efficacy were dynamically monitored through BLT. The results in this study showed that the growth of liver cancer can be dynamically monitored from very early stage, and also the sorafenib treatment efficacy can be reliably and objectively assessed using BLT imaging method. Our experimental result demonstrated sorafenib can inhibit the tumor growth effectively. BLT enabled the non-invasive and reliable assessment of anti-neoplastic drug efficacy on liver cancer.

Using bacterial bioluminescence to evaluate the impact of biofilm on porous media hydraulic properties.

PubMed

Bozorg, Ali; Gates, Ian D; Sen, Arindom

2015-02-01

Biofilm formation in natural and engineered porous systems can significantly impact hydrodynamics by reducing porosity and permeability. To better understand and characterize how biofilms influence hydrodynamic properties in porous systems, the genetically engineered bioluminescent bacterial strain Pseudomonas fluorescens HK44 was used to quantify microbial population characteristics and biofilm properties in a translucent porous medium. Power law relationships were found to exist between bacterial bioluminescence and cell density, fraction of void space occupied by biofilm (i.e. biofilm saturation), and hydraulic conductivity. The simultaneous evaluation of biofilm saturation and porous medium hydraulic conductivity in real time using a non-destructive approach enabled the construction of relative hydraulic conductivity curves. Such information can facilitate simulation studies related to biological activity in porous structures, and support the development of new models to describe the dynamic behavior of biofilm and fluid flow in porous media. The bioluminescence based approach described here will allow for improved understanding and control of industrially relevant processes such as biofiltration and bioremediation. Copyright © 2014. Published by Elsevier B.V.

Molecular Origin of Color Variation in Firefly (Beetle) Bioluminescence: A Chemical Basis for Biological Imaging.

PubMed

Hirano, Takashi

2016-01-01

Firefly shows bioluminescence by "luciferin-luciferase" (L-L) reaction using luciferin, luciferase, ATP and O2. The chemical photon generation by an enzymatic reaction is widely utilized for analytical methods including biological imaging in the life science fields. To expand photondetecting analyses with firefly bioluminescence, it is important for users to understand the chemical basis of the L-L reaction. In particular, the emission color variation of the L-L reaction is one of the distinguishing characteristics for multicolor luciferase assay and in vivo imaging. From the viewpoint of fundamental chemistry, this review explains the recent progress in the studies on the molecular mechanism of emission color variation after showing the outline of the reaction mechanism of the whole L-L reaction. On the basis of the mechanism, the progresses in organic synthesis of luciferin analogs modulating their emission colors are also presented to support further developments of red/near infrared in vivo biological imaging utility of firefly bioluminescence.

Local gene transfection in the cochlea (Review).

PubMed

Xia, Li; Yin, Shankai

2013-07-01

There is much interest in the potential application of vector-induced gene therapeutic approaches to several forms of hearing disorders due to the poor efficacy of existing treatments. The cochlea is an ideal site for local gene transfection due to its anatomical encapsulation and fluid flow within its ducts. However, this requires the development of novel technologies in materials science and microbial supply vectors for target gene delivery. This review focuses on the introduction of various viral and non-viral vectors as well as injection approaches to transfecting cochlear cells in vivo. Finally, the perspective of local gene therapy was discussed. Therapeutic approaches using local gene transfection may provide a means of cochlear cell and tissue protection and treatment in cases of exogenous hearing loss and endogenous disorders.

A Bioluminescent Whole-Cell Reporter for Detection of 2,4-Dichlorophenoxyacetic Acid and 2,4-Dichlorophenol in Soil

PubMed Central

Hay, Anthony G.; Rice, James F.; Applegate, Bruce M.; Bright, Nathan G.; Sayler, Gary S.

2000-01-01

A bioreporter was made containing a tfdRPDII-luxCDABE fusion in a modified mini-Tn5 construct. When it was introduced into the chromosome of Ralstonia eutropha JMP134, the resulting strain, JMP134-32, produced a sensitive bioluminescent response to 2,4-dichlorophenoxyacetic acid (2,4-D) at concentrations of 2.0 μM to 5.0 mM. This response was linear (R2 = 0.9825) in the range of 2.0 μM to 1.1 × 102 μM. Saturation occurred at higher concentrations, with maximal bioluminescence occurring in the presence of approximately 1.2 mM 2,4-D. A sensitive response was also recorded in the presence of 2,4-dichlorophenol at concentrations below 1.1 × 102 μM; however, only a limited bioluminescent response was recorded in the presence of 3-chlorobenzoic acid at concentrations below 1.0 mM. A significant bioluminescent response was also recorded when strain JMP134-32 was incubated with soils containing aged 2,4-D residues. PMID:11010925

Gene Transfection Method Using Atmospheric Pressure Dielectric-Barrier Discharge Plasmas

NASA Astrophysics Data System (ADS)

Sasaki, Shota; Kanzaki, Makoto; Kaneko, Toshiro

2013-09-01

Gene transfection which is the process of deliberately introducing nucleic acids into cells is expected to play an important role in medical treatment because the process is necessary for gene therapy and creation of induced pluripotent stem (iPS) cells. However, the conventional transfection methods have some problems, so we focus attention on promising transfection methods by atmospheric pressure dielectric-barrier discharge (AP-DBD) plasmas. AP-DBD He plasmas are irradiated to the living cell covered with genes. Preliminarily, we use fluorescent dye YOYO-1 instead of the genes and use LIVE/DEAD Stain for cell viability test, and we analyze the transfection efficiency and cell viability under the various conditions. It is clarified that the transfection efficiency is strongly dependence on the plasma irradiation time and cell viability rates is high rates (>90%) regardless of long plasma irradiation time. These results suggest that ROS (Reactive Oxygen Species) and electric field generated by the plasma affect the gene transfection. In addition to this (the plasma irradiation time) dependency, we now investigate the effect of the plasma irradiation under the various conditions.

Comparison of bioluminescent kinase assays using substrate depletion and product formation.

PubMed

Tanega, Cordelle; Shen, Min; Mott, Bryan T; Thomas, Craig J; MacArthur, Ryan; Inglese, James; Auld, Douglas S

2009-12-01

Assays for ATPases have been enabled for high-throughput screening (HTS) by employing firefly luciferase to detect the remaining ATP in the assay. However, for any enzyme assay, measurement of product formation is a more sensitive assay design. Recently, technologies that allow detection of the ADP product from ATPase reactions have been described using fluorescent methods of detection. We describe here the characterization of a bioluminescent assay that employs firefly luciferase in a coupled-enzyme assay format to enable detection of ADP levels from ATPase assays (ADP-Glo, Promega Corp.). We determined the performance of the ADP-Glo assay in 1,536-well microtiter plates using the protein kinase Clk4 and a 1,352 member kinase focused combinatorial library. The ADP-Glo assay was compared to the Clk4 assay performed using a bioluminescence ATP-depletion format (Kinase-Glo, Promega Corp). We performed this analysis using quantitative HTS (qHTS) where we determined potency values for all library members and identified approximately 300 compounds with potencies ranging from as low as 50 nM to >10 microM, yielding a robust dataset for the comparison. Both assay formats showed high performance (Z'-factors approximately 0.9) and showed a similar potency distribution for the actives. We conclude that the bioluminescence ADP detection assay system is a viable generic alternative to the widely used ATP-depletion assay for ATPases and discuss the advantages and disadvantages of both approaches.

Development of bacteriophage-based bioluminescent bioreporters for monitoring of microbial pathogens

NASA Astrophysics Data System (ADS)

Ozen, Aysu; Montgomery, Kacey; Jegier, Pat; Patterson, Stacey; Daumer, Kathleen A.; Ripp, Steven A.; Garland, Jay L.; Sayler, Gary S.

2004-03-01

Microorganisms pose numerous problems when present in human occupied enclosed environments. Primary among these are health related hazards, manifested as infectious diseases related to contaminated drinking water, food, or air circulation systems or non-infectious allergy related complications associated with microbial metabolites (sick building syndrome). As a means towards rapid detection of microbial pathogens, we are attempting to harness the specificity of bacterial phage for their host with a modified quorum sensing amplification signal to produce quantifiable bioluminescent (lux) detection on a silicon microluminometer. The bacteriophage itself is metabolically inactive, only achieving replicative capabilities upon infection of its specific host bacterium. Bacteriophage bioluminescent bioreporters contain a genomically inserted luxI component. During an infection event, the phage genes and accompanying luxI construct are taken up by the host bacterium and transcribed, resulting in luxI expression and subsequent activation of a homoserine lactone inducible bioluminescent bioreporter. We constructed a vector carrying the luxI gene under the control of a strong E. coli promoter and cloned it into E. coli. We have shown that it can induce luminescence up to 14,000 counts per second when combined with the bioreporter strain. In their final embodiment, these sensors will be fully independent microelectronic monitors for microbial contamination, requiring only exposure of the biochip to the sample, with on-chip signal processing downloaded directly to the local area network of the environmental control system.

ATP bioluminescence: Surface hygiene monitoring in milk preparation room of neonatal intensive care unit

NASA Astrophysics Data System (ADS)

Mohamad, Mahirah; Ishak, Shareena; Jaafar, Rohana; Sani, Norrakiah Abdullah

2018-04-01

ATP Bioluminescence application and standard microbiological analyses were used to evaluate the cleanliness of milk contact surfaces and non-milk contact surfaces in milk preparation room of neonatal intensive care unit (NICU) of Universiti Kebangsaan Malaysia Medical Centre (UKMMC). A total of 44 samples including the breast pump, milk bottle, milk bottle screw top and screw ring, teats, measuring cups, waterless warmer, refrigerator, dishwasher and pasteurizer inner wall were tested on May 2017. 3M Clean and Trace Hygiene Monitoring (UXL100 ATP Test swabs) and the bioluminescence reader Clean-Trace NG Luminometer (3M) were used to measure the Relative Light Unit (RLU) and microbiological analysis using 3M Quick Swab and 3MTM PetrifilmTM for enumeration of aerobic count, Staphylococcus aureus, Enterobacteriaceae, coliform and detection of Escherichia coli (CFU /100cm2 or utensil/item). The RLU values were from 11 to 194 and passed the ATP benchmark for intensive care unit (ICU), < 250 RLU as recommended. Aerobic colony count was only found in waterless warmer (0.05±0.01 mean log CFU/warmer). None of S. aureus, Enterobacteriaceae, E. coli and coliform was detected in all samples. A weak correlation was found between bioluminescence measurements RLU and the microbiological analysis (CFU). However, the use of ATP bioluminescence in monitoring milk preparation room cleanliness can be a useful method for assessing rapidly the surface hygiene as well as to verify the Sanitation Standard Operating Procedure (SSOP) prior to implementation of Hazard Analysis and Critical Control Points (HACCP) in milk preparation room.

Recombination and Transfection Mapping of Cistron 5 of Bacteriophage Sp82g

PubMed Central

Green, D. MacDonald; Urban, Margeret I.

1972-01-01

Recombination between transfecting SP82G DNA molecules has been studied in Bacillus subtilis. Recombinant progeny issuing from transfected cells show many of the features that characterize progeny production in multiplicity reactivated bacteriophage, such as: majority recombinant clones, non-reciprocity of recombinant clones and the frequent absence of input alleles. While transfection substantially lowers the linkage observed between markers in normal phage crosses, linkage is observed at small map distances in transfection either by plating transfected bacteria or the progeny phage. Maps constructed from transfection crosses are identical to those of normal phage crosses, except in magnitude.—Examination of the concentration response of two marker biparental crosses, and three marker triparental crosses using transfecting DNA leads to the conclusion that at all concentrations, transfective centers are saturated with respect to the number of molecules that can be taken up. Thus, the frequency of recombinant infective centers, or recombinant progeny is independent of concentration effects. PMID:17248556

Bio-Orthogonal Mediated Nucleic Acid Transfection of Cells via Cell Surface Engineering.

PubMed

O'Brien, Paul J; Elahipanah, Sina; Rogozhnikov, Dmitry; Yousaf, Muhammad N

2017-05-24

The efficient delivery of foreign nucleic acids (transfection) into cells is a critical tool for fundamental biomedical research and a pillar of several biotechnology industries. There are currently three main strategies for transfection including reagent, instrument, and viral based methods. Each technology has significantly advanced cell transfection; however, reagent based methods have captured the majority of the transfection market due to their relatively low cost and ease of use. This general method relies on the efficient packaging of a reagent with nucleic acids to form a stable complex that is subsequently associated and delivered to cells via nonspecific electrostatic targeting. Reagent transfection methods generally use various polyamine cationic type molecules to condense with negatively charged nucleic acids into a highly positively charged complex, which is subsequently delivered to negatively charged cells in culture for association, internalization, release, and expression. Although this appears to be a straightforward procedure, there are several major issues including toxicity, low efficiency, sorting of viable transfected from nontransfected cells, and limited scope of transfectable cell types. Herein, we report a new strategy (SnapFect) for nucleic acid transfection to cells that does not rely on electrostatic interactions but instead uses an integrated approach combining bio-orthogonal liposome fusion, click chemistry, and cell surface engineering. We show that a target cell population is rapidly and efficiently engineered to present a bio-orthogonal functional group on its cell surface through nanoparticle liposome delivery and fusion. A complementary bio-orthogonal nucleic acid complex is then formed and delivered to which chemoselective click chemistry induced transfection occurs to the primed cell. This new strategy requires minimal time, steps, and reagents and leads to superior transfection results for a broad range of cell types

PLUME DISPERSION IN STABLY STRATIFIED FLOWS OVER COMPLEX TERRAIN, PHASE 2

EPA Science Inventory

Laboratory experiments were conducted in a stratified towing tank to investigate plume dispersion in stably stratified flows. First, plume dispersion over an idealized terrain model with a simulated elevated inversion in the atmosphere was investigated. These results were compare...

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

PubMed Central

Deshpande, Mangesh C.; Prausnitz, Mark R.

2007-01-01

Ultrasound and poly(ethylenimine) (PEI) have each separately been shown to increase DNA transfection efficiency. This study tested the hypothesis that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. This in vitro study assessed transfection efficiency of two different DNA plasmids encoding green fluorescent protein and firefly luciferase in two different cells types, a primary culture of human aortic smooth muscle cells and an immortal line of human prostrate cancer cells. We found that ultrasound sonication increased transfection up to 18-fold, DNA complexation with PEI increased transfection up to 90-fold, and the combination of ultrasound and PEI synergistically increased transfection up to 200-fold, which resulted in reporter gene expression by 34% of cells. Kinetic measurements found that the effects of ultrasound alone acted quickly, whereas increased transfection by PEI either alone or in combination with ultrasound strongly benefited from a 4-h incubation with the DNA plasmid after sonication. Although serum reduced absolute expression levels, it did not affect the relative increase in transfection when ultrasound was added to PEI enhancement. Flow cytometry measurements showed that sonication increased intracellular uptake of labelled DNA complexed to PEI by 55% relative to PEI complexation alone. Electrophoresis assay showed no damage to DNA or PEI-DNA complexes after sonication. Overall, these results suggest that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. PMID:17258835

A reporter gene assay for the detection of phytoestrogens in traditional Chinese medicine.

PubMed

Miller-Martini, D M; Chan, R Y; Ip, N Y; Sheu, S J; Wong, Y H

2001-09-01

Bupleurum & Peony Formula (Jia Wei Xiao Yao San) is a herbal formula which possesses a clinical history for the treatment of menopausal syndrome and menstrual irregularity. The present investigation reports the ability to monitor the formula's phytoestrogen content that will allow for the implementation of a standardization protocol that is based on a quantifiable biological response. Utilizing an oestrogen-sensitive chimeric receptor/reporter gene element which has been stably transfected into HeLa cells, the botanical formula was shown to induce the expression of the reporter gene, luciferase, in a dose dependent manner. Pretreatment of the HeLa cells with the botanical formula produced a 5-fold increase in bioluminescence compared with the control. Additionally, our studies showed that the response of the cells, when challenged by the botanical formula, was oestrogen specific. Pretreatment of the cells with tamoxifen effectively blocked the activation of the chimeric oestrogen receptor by the botanical formula. The cell line provides a sensitive assay that can easily detect the presence of phytoestrogens in complex botanical formulas. Copyright 2001 John Wiley & Sons, Ltd.

Hepa1-6-FLuc cell line with the stable expression of firefly luciferase retains its primary properties with promising bioluminescence imaging ability.

PubMed

Li, Yasha; Liu, Mengnan; Cui, Jiejie; Yang, Ke; Zhao, Li; Gong, Mengjia; Wang, Yi; He, Yun; He, Tongchuan; Bi, Yang

2018-05-01

Reliable animal models are required for the in vivo study of the molecular mechanisms and effects of chemotherapeutic drugs in hepatocarcinoma. In vivo tracing techniques based on firefly luciferase (FLuc) may optimize the non-invasive monitoring of experimental animals. The present study established a murine Hepa1-6-FLuc cell line that stably expressed a retrovirus-delivered FLuc protein gene. The cell morphology, proliferation, migration and invasion ability of Hepa1-6-FLuc cells were the same as that of the Hepa1-6 cells, and thus is suitable to replace Hepa1-6 cells in the construction of hepatocarcinoma animal models. No differences in subcutaneous tumor mass and its pathomorphology from implanted Hepa1-6-FLuc cells were observed compared with Hepa1-6 control tumors. Bioluminescence imaging indicated that the Luc signal of the Hepa1-6-FLuc cells was consistently strengthened with increases in tumor mass; however, the Luc signal of Hepa1-6-AdFLuc became weaker and eventually disappeared during tumor development. Therefore, compared with the transient expression by adenovirus, stable expression of the FLuc gene in Hepa1-6 cells may better reflect cell proliferation and survival in vivo , and provide a reliable source for the establishment of hepatocarcinoma models.

Bioluminescence imaging of Clavibacter michiganensis subsp. michiganensis infection of tomato seeds and plants.

PubMed

Xu, Xiulan; Miller, Sally A; Baysal-Gurel, Fulya; Gartemann, Karl-Heinz; Eichenlaub, Rudolf; Rajashekara, Gireesh

2010-06-01

Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the

Development of a rapid optic bacteria detecting system based on ATP bioluminescence

NASA Astrophysics Data System (ADS)

Liu, Jun Tao; Luo, JinPing; Liu, XiaoHong; Cai, XinXia

2014-12-01

A rapid optic bacteria detecting system based on the principle of Adenosine triphosphate(ATP) bioluminescence was presented in this paper. This system consisted of bioluminescence-based biosensor and the high-sensitivity optic meter. A photon counting photomultiplier tube (PMT) module was used to improve the detection sensitivity, and a NIOS II/f processor based on a Field Programmable Gate Array(FPGA) was used to control the system. In this work, Micrococcus luteus were chosen as the test sample. Several Micrococcus luteus suspension with different concentration was tested by both T2011 and plate counting method. By comparing the two group results, an calibration curve was obtained from the bioluminescence intensity for Micrococcus luteus in the range of 2.3×102 ~ 2.3×106 CFU/mL with a good correlation coefficient of 0.960. An impacting Air microorganism sampler was used to capture Airborne Bacteria, and 8 samples were collected in different place. The TBC results of 8 samples by T2011 were between 10 ~ 2×103 cfu/mL, consistent with that of plate counting method, which indicated that 8 samples were between 10 ~ 3×103 cfu/mL. For total airborne bacteria count was small, correlation coefficient was poor. Also no significant difference was found between T2011 and plate counting method by statistical analyses.

[Determination of minimal concentrations of biocorrosion inhibitors by a bioluminescence method in relation to bacteria, participating in biocorrosion].

PubMed

Efremenko, E N; Azizov, R E; Makhlis, T A; Abbasov, V M; Varfolomeev, S D

2005-01-01

By using a bioluminescence ATP assay, we have determined the minimal concentrations of some biocorrosion inhibitors (Katon, Khazar, VFIKS-82, Nitro-1, Kaspii-2, and Kaspii-4) suppressing most common microbial corrosion agents: Desulfovibrio desulfuricans, Desulfovibrio vulgaris, Pseudomonas putida, Pseudomonas fluorescens, and Acidithiobacillus ferrooxidans. The cell titers determined by the bioluminescence method, including not only dividing cells but also their dormant living counterparts, are two- to sixfold greater than the values determined microbiologically. It is shown that the bioluminescence method can be applied to determination of cell titers in samples of oil-field waters in the presence of iron ions (up to 260 mM) and iron sulfide (to 186 mg/l) and in the absence or presence of biocidal corrosion inhibitors.

Inertial bioluminescence rhythms at the Capo Passero (KM3NeT-Italia) site, Central Mediterranean Sea

NASA Astrophysics Data System (ADS)

Aguzzi, J.; Fanelli, E.; Ciuffardi, T.; Schirone, A.; Craig, J.; Aiello, S.; Ameli, F.; Anghinolfi, M.; Barbarino, G.; Barbarito, E.; Beverini, N.; Biagi, S.; Biagioni, A.; Bouhadef, B.; Bozza, C.; Cacopardo, G.; Calamai, M.; Calì, C.; Capone, A.; Caruso, F.; Cecchini, S.; Ceres, A.; Chiarusi, T.; Circella, M.; Cocimano, R.; Coniglione, R.; Costa, M.; Cuttone, G.; D'Amato, C.; D'Amico, A.; de Bonis, G.; de Luca, V.; Deniskina, N.; Distefano, C.; di Mauro, L. S.; Fermani, P.; Ferrara, G.; Flaminio, V.; Fusco, L. A.; Garufi, F.; Giordano, V.; Gmerk, A.; Grasso, R.; Grella, G.; Hugon, C.; Imbesi, M.; Kulikovskiy, V.; Larosa, G.; Lattuada, D.; Leismüller, K. P.; Leonora, E.; Litrico, P.; Lonardo, A.; Longhitano, F.; Presti, D. Lo; Maccioni, E.; Margiotta, A.; Marinelli, A.; Martini, A.; Masullo, R.; Mele, R.; Migliozzi, P.; Migneco, E.; Miraglia, A.; Mollo, C. M.; Mongelli, M.; Morganti, M.; Musico, P.; Musumeci, M.; Nicolau, C. A.; Orlando, A.; Orzelli, A.; Papaleo, R.; Pellegrino, C.; Pellegriti, M. G.; Perrina, C.; Piattelli, P.; Poma, E.; Pulvirenti, S.; Raffaelli, F.; Randazzo, N.; Riccobene, G.; Rovelli, A.; Sanguineti, M.; Sapienza, P.; Sciacca, V.; Sgura, I.; Simeone, F.; Sipala, V.; Speziale, F.; Spitaleri, A.; Spurio, M.; Stellacci, S. M.; Taiuti, M.; Terreni, G.; Trasatti, L.; Trovato, A.; Versari, F.; Vicini, P.; Viola, S.; Vivolo, D.

2017-03-01

In the deep sea, the sense of time is dependent on geophysical fluctuations, such as internal tides and atmospheric-related inertial currents, rather than day-night rhythms. Deep-sea neutrino telescopes instrumented with light detecting Photo-Multiplier Tubes (PMT) can be used to describe the synchronization of bioluminescent activity of abyssopelagic organisms with hydrodynamic cycles. PMT readings at 8 different depths (from 3069 to 3349 m) of the NEMO Phase 2 prototype, deployed offshore Capo Passero (Sicily) at the KM3NeT-Italia site, were used to characterize rhythmic bioluminescence patterns in June 2013, in response to water mass movements. We found a significant (p < 0.05) 20.5 h periodicity in the bioluminescence signal, corresponding to inertial fluctuations. Waveform and Fourier analyses of PMT data and tower orientation were carried out to identify phases (i.e. the timing of peaks) by subdividing time series on the length of detected inertial periodicity. A phase overlap between rhythms and cycles suggests a mechanical stimulation of bioluminescence, as organisms carried by currents collide with the telescope infrastructure, resulting in the emission of light. A bathymetric shift in PMT phases indicated that organisms travelled in discontinuous deep-sea undular vortices consisting of chains of inertially pulsating mesoscale cyclones/anticyclones, which to date remain poorly known.

Inertial bioluminescence rhythms at the Capo Passero (KM3NeT-Italia) site, Central Mediterranean Sea

PubMed Central

Aguzzi, J.; Fanelli, E.; Ciuffardi, T.; Schirone, A.; Craig, J.; Aiello, S.; Ameli, F.; Anghinolfi, M.; Barbarino, G.; Barbarito, E.; Beverini, N.; Biagi, S.; Biagioni, A.; Bouhadef, B.; Bozza, C.; Cacopardo, G.; Calamai, M.; Calì, C.; Capone, A.; Caruso, F.; Cecchini, S.; Ceres, A.; Chiarusi, T.; Circella, M.; Cocimano, R.; Coniglione, R.; Costa, M.; Cuttone, G.; D’Amato, C.; D’Amico, A.; De Bonis, G.; De Luca, V.; Deniskina, N.; Distefano, C.; Di Mauro, L. S.; Fermani, P.; Ferrara, G.; Flaminio, V.; Fusco, L. A.; Garufi, F.; Giordano, V.; Gmerk, A.; Grasso, R.; Grella, G.; Hugon, C.; Imbesi, M.; Kulikovskiy, V.; Larosa, G.; Lattuada, D.; Leismüller, K. P.; Leonora, E.; Litrico, P.; Lonardo, A.; Longhitano, F.; Presti, D. Lo; Maccioni, E.; Margiotta, A.; Marinelli, A.; Martini, A.; Masullo, R.; Mele, R.; Migliozzi, P.; Migneco, E.; Miraglia, A.; Mollo, C. M.; Mongelli, M.; Morganti, M.; Musico, P.; Musumeci, M.; Nicolau, C. A.; Orlando, A.; Orzelli, A.; Papaleo, R.; Pellegrino, C.; Pellegriti, M. G.; Perrina, C.; Piattelli, P.; Poma, E.; Pulvirenti, S.; Raffaelli, F.; Randazzo, N.; Riccobene, G.; Rovelli, A.; Sanguineti, M.; Sapienza, P.; Sciacca, V.; Sgura, I.; Simeone, F.; Sipala, V.; Speziale, F.; Spitaleri, A.; Spurio, M.; Stellacci, S. M.; Taiuti, M.; Terreni, G.; Trasatti, L.; Trovato, A.; Versari, F.; Vicini, P.; Viola, S.; Vivolo, D.

2017-01-01

In the deep sea, the sense of time is dependent on geophysical fluctuations, such as internal tides and atmospheric-related inertial currents, rather than day-night rhythms. Deep-sea neutrino telescopes instrumented with light detecting Photo-Multiplier Tubes (PMT) can be used to describe the synchronization of bioluminescent activity of abyssopelagic organisms with hydrodynamic cycles. PMT readings at 8 different depths (from 3069 to 3349 m) of the NEMO Phase 2 prototype, deployed offshore Capo Passero (Sicily) at the KM3NeT-Italia site, were used to characterize rhythmic bioluminescence patterns in June 2013, in response to water mass movements. We found a significant (p < 0.05) 20.5 h periodicity in the bioluminescence signal, corresponding to inertial fluctuations. Waveform and Fourier analyses of PMT data and tower orientation were carried out to identify phases (i.e. the timing of peaks) by subdividing time series on the length of detected inertial periodicity. A phase overlap between rhythms and cycles suggests a mechanical stimulation of bioluminescence, as organisms carried by currents collide with the telescope infrastructure, resulting in the emission of light. A bathymetric shift in PMT phases indicated that organisms travelled in discontinuous deep-sea undular vortices consisting of chains of inertially pulsating mesoscale cyclones/anticyclones, which to date remain poorly known. PMID:28332561

PC5-A-mediated processing of pro-neurotensin in early compartments of the regulated secretory pathway of PC5-transfected PC12 cells.

PubMed

Barbero, P; Rovère, C; De Bie, I; Seidah, N; Beaudet, A; Kitabgi, P

1998-09-25

Among the members of the proprotein convertase (PC) family, PC1 and PC2 have well established roles as prohormone convertases. Another good candidate for this role is PC5-A that has been shown to be present in the regulated secretory pathway of certain neuroendocrine tissues, but evidence that it can process prohormones is lacking. To determine whether PC5-A could function as a prohormone convertase and to compare its cleavage specificity with that of PC1 and PC2, we stably transfected the rat pheochromocytoma PC12 cell line with PC5-A and analyzed the biosynthesis and subcellular localization of the enzyme, as well as its ability to process pro-neurotensin/neuromedin N (pro-NT/NN) into active peptides. Our data showed that in transfected PC12 cells, PC5-A was converted from its 126-kDa precursor form into a 117-kDa mature form and, to a lesser extent, into a C-terminally truncated 65-kDa form of the 117-kDa product. Metabolic and immunochemical studies showed that PC5-A was sorted to early compartments of the regulated secretory pathway where it colocalized with immunoreactive NT. Furthermore, pro-NT/NN was processed in these compartments according to a pattern that differed from that previously described in PC1- and PC2-transfected PC12 cells. This pattern resembled that previously reported for pro-NT/NN processing in the adrenal medulla, a tissue known to express high levels of PC5-A. Altogether, these data demonstrate for the first time the ability of PC5-A to function as a prohormone convertase in the regulated secretory pathway and suggest a role for this enzyme in the physiological processing of pro-NT/NN.

Monitoring Bloom Dynamics of a Common Coastal Bioluminescent Ctenophore

DTIC Science & Technology

2009-09-30

potential in the coastal zone environment. OBJECTIVES Blooms of bioluminescent jellyfish , especially of Mnemiopsis leidyi, are a common occurrence... jellyfish populations are done with net collections by hand at stations weekly, monthly, or seasonally. These time scales severely limit our knowledge...the collection of both biotic and abiotic data continuously. 5 IMPACT/APPLICATIONS As incidents of jellyfish blooms, especially Mnemiopsis

Sensitive in situ monitoring of a recombinant bioluminescent Yersinia enterocolitica reporter mutant in real time on Camembert cheese.

PubMed

Maoz, Ariel; Mayr, Ralf; Bresolin, Geraldine; Neuhaus, Klaus; Francis, Kevin P; Scherer, Siegfried

2002-11-01

Bioluminescent mutants of Yersinia enterocolitica were generated by transposon mutagenesis using a promoterless, complete lux operon (luxCDABE) derived from Photorhabdus luminescens, and their production of light in the cheese environment was monitored. Mutant B94, which had the lux cassette inserted into an open reading frame of unknown function was used for direct monitoring of Y. enterocolitica cells on cheeses stored at 10 degrees C by quantifying bioluminescence using a photon-counting, intensified charge-coupled device camera. The detection limit on cheese was 200 CFU/cm(2). Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, and cheese), as well as by growth phase, via the promoter the lux operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde in the food matrix in order to gain quantitative, reproducible bioluminescence data. As far as we know, this is the first time a pathogen has been monitored in situ, in real time, in a "real-product" status, and at a low temperature.

Sensitive In Situ Monitoring of a Recombinant Bioluminescent Yersinia enterocolitica Reporter Mutant in Real Time on Camembert Cheese

PubMed Central

Maoz, Ariel; Mayr, Ralf; Bresolin, Geraldine; Neuhaus, Klaus; Francis, Kevin P.; Scherer, Siegfried

2002-01-01

Bioluminescent mutants of Yersinia enterocolitica were generated by transposon mutagenesis using a promoterless, complete lux operon (luxCDABE) derived from Photorhabdus luminescens, and their production of light in the cheese environment was monitored. Mutant B94, which had the lux cassette inserted into an open reading frame of unknown function was used for direct monitoring of Y. enterocolitica cells on cheeses stored at 10°C by quantifying bioluminescence using a photon-counting, intensified charge-coupled device camera. The detection limit on cheese was 200 CFU/cm2. Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, and cheese), as well as by growth phase, via the promoter the lux operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde in the food matrix in order to gain quantitative, reproducible bioluminescence data. As far as we know, this is the first time a pathogen has been monitored in situ, in real time, in a “real-product” status, and at a low temperature. PMID:12406772

In vivo bioluminescence tomography based on multi-view projection and 3D surface reconstruction

NASA Astrophysics Data System (ADS)

Zhang, Shuang; Wang, Kun; Leng, Chengcai; Deng, Kexin; Hu, Yifang; Tian, Jie

2015-03-01

Bioluminescence tomography (BLT) is a powerful optical molecular imaging modality, which enables non-invasive realtime in vivo imaging as well as 3D quantitative analysis in preclinical studies. In order to solve the inverse problem and reconstruct inner light sources accurately, the prior structural information is commonly necessary and obtained from computed tomography or magnetic resonance imaging. This strategy requires expensive hybrid imaging system, complicated operation protocol and possible involvement of ionizing radiation. The overall robustness highly depends on the fusion accuracy between the optical and structural information. In this study we present a pure optical bioluminescence tomographic system (POBTS) and a novel BLT method based on multi-view projection acquisition and 3D surface reconstruction. The POBTS acquired a sparse set of white light surface images and bioluminescent images of a mouse. Then the white light images were applied to an approximate surface model to generate a high quality textured 3D surface reconstruction of the mouse. After that we integrated multi-view luminescent images based on the previous reconstruction, and applied an algorithm to calibrate and quantify the surface luminescent flux in 3D.Finally, the internal bioluminescence source reconstruction was achieved with this prior information. A BALB/C mouse with breast tumor of 4T1-fLuc cells mouse model were used to evaluate the performance of the new system and technique. Compared with the conventional hybrid optical-CT approach using the same inverse reconstruction method, the reconstruction accuracy of this technique was improved. The distance error between the actual and reconstructed internal source was decreased by 0.184 mm.

Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters.

PubMed

Rogers, Kelly L; Stinnakre, Jacques; Agulhon, Cendra; Jublot, Delphine; Shorte, Spencer L; Kremer, Eric J; Brûlet, Philippe

2005-02-01

Measurements of local Ca2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto-fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution. Aequorin is a genetically encoded Ca(2+)-sensitive bioluminescent protein, however, its low quantum yield prevents dynamic measurements of Ca2+ responses in single cells. To overcome this limitation, we recently reported the bi-functional Ca2+ reporter gene, GFP-aequorin (GA), which was developed specifically to improve the light output and stability of aequorin chimeras [V. Baubet, et al., (2000) PNAS, 97, 7260-7265]. In the current study, we have genetically targeted GA to different microdomains important in synaptic transmission, including to the mitochondrial matrix, endoplasmic reticulum, synaptic vesicles and to the postsynaptic density. We demonstrate that these reporters enable 'real-time' measurements of subcellular Ca2+ changes in single mammalian neurons using bioluminescence. The high signal-to-noise ratio of these reporters is also important in that it affords the visualization of Ca2+ dynamics in cell-cell communication in neuronal cultures and tissue slices. Further, we demonstrate the utility of this approach in ex-vivo preparations of mammalian retina, a paradigm in which external light input should be controlled. This represents a novel molecular imaging approach for non-invasive monitoring of local Ca2+ dynamics and cellular communication in tissue or whole animal studies.

Sensitivity of the Geomagnetic Octupole to a Stably Stratified Layer in the Earth's Core

NASA Astrophysics Data System (ADS)

Yan, C.; Stanley, S.

2017-12-01

The presence of a stably stratified layer at the top of the core has long been proposed for Earth, based on evidence from seismology and geomagnetic secular variation. Geodynamo modeling offers a unique window to inspect the properties and dynamics in Earth's core. For example, numerical simulations have shown that magnetic field morphology is sensitive to the presence of stably stratified layers in a planet's core. Here we use the mMoSST numerical dynamo model to investigate the effects of a thin stably stratified layer at the top of the fluid outer core in Earth on the resulting large-scale geomagnetic field morphology. We find that the existence of a stable layer has significant influence on the octupolar component of the magnetic field in our models, whereas the quadrupole doesn't show an obvious trend. This suggests that observations of the geomagnetic field can be applied to provide information of the properties of this plausible stable layer, such as how thick and how stable this layer could be. Furthermore, we have examined whether the dominant thermal signature from mantle tomography at the core-mantle boundary (CMB) (a degree & order 2 spherical harmonic) can influence our results. We found that this heat flux pattern at the CMB has no outstanding effects on the quadrupole and octupole magnetic field components. Our studies suggest that if there is a stably stratified layer at the top of the Earth's core, it must be limited in terms of stability and thickness, in order to be compatible with the observed paleomagnetic record.

Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity.

PubMed

Cevenini, Luca; Calabretta, Maria Maddalena; Lopreside, Antonia; Tarantino, Giuseppe; Tassoni, Annalisa; Ferri, Maura; Roda, Aldo; Michelini, Elisa

2016-12-01

The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products. Graphical abstract Smartphone-based bioluminescence cell biosensor.

Aequorin fusion proteins as bioluminescent tracers for competitive immunoassays

NASA Astrophysics Data System (ADS)

Mirasoli, Mara; Michelini, Elisa; Deo, Sapna K.; Dikici, Emre; Roda, Aldo; Daunert, Sylvia

2004-06-01

The use of bio- and chemiluminescence for the development of quantitative binding assays offers undoubted advantages over other detection systems, such as spectrophotometry, fluorescence, or radioactivity. Indeed, bio- and chemiluminescence detection provides similar, or even better, sensitivity and detectability than radioisotopes, while avoiding the problems of health hazards, waste disposal, and instability associated with the use of radioisotopes. Among bioluminescent labels, the calcium-activated photoprotein aequorin, originally isolated from Aequorea victoria and today available as a recombinant product, is characterized by very high detectability, down to attomole levels. It has been used as a bioluminescent label for developing a variety of highly sensitive immunoassays, using various analyte-aequorin conjugation strategies. When the analyte is a protein or a peptide, genetic engineering techniques can be used to produce protein fusions where the analyte is in-frame fused with aequorin, thus producing homogeneous one-to-one conjugation products, available in virtually unlimited amount. Various assays were developed using this strategy: a short review of the most interesting applications is presented, as well as the cloning, purification and initial characterization of an endothelin-1-aequorin conjugate suitable for developing a competitive immunoassay for endothelin-1, a potent vasoconstrictor peptide, involved in hypertension.

Structure-activity correlation in transfection promoted by pyridinium cationic lipids.

PubMed

Parvizi-Bahktar, P; Mendez-Campos, J; Raju, L; Khalique, N A; Jubeli, E; Larsen, H; Nicholson, D; Pungente, M D; Fyles, T M

2016-03-21

The efficiency of the transfection of a plasmid DNA encoding a galactosidase promoted by a series of pyridinium lipids in mixtures with other cationic lipids and neutral lipids was assessed in CHO-K1 cells. We identify key molecular parameters of the lipids in the mixture - clog P, lipid length, partial molar volume - to predict the morphology of the lipid-DNA lipoplex and then correlate these same parameters with transfection efficiency in an in vitro assay. We define a Transfection Index that provides a linear correlation with normalized transfection efficiency over a series of 90 different lipoplex compositions. We also explore the influence of the same set of molecular parameters on the cytotoxicity of the formulations.

A genetic screen for bioluminescence genes in the fungus Armillaria mellea, through the use of Agrobacterium tumefaciens-mediated random insertional mutagenesis

USDA-ARS?s Scientific Manuscript database

Bioluminescence is reported from 71 saprobic species of fungi from four, distant lineages in the order Agaricales. Analyses of the fungal luminescent chemistry shows that all four lineages share a functionally conserved substrate and luciferase, indicating that the bioluminescent pathway is likely c...

Single-particle dispersion in stably stratified turbulence

NASA Astrophysics Data System (ADS)

Sujovolsky, N. E.; Mininni, P. D.; Rast, M. P.

2018-03-01

We present models for single-particle dispersion in vertical and horizontal directions of stably stratified flows. The model in the vertical direction is based on the observed Lagrangian spectrum of the vertical velocity, while the model in the horizontal direction is a combination of a continuous-time eddy-constrained random walk process with a contribution to transport from horizontal winds. Transport at times larger than the Lagrangian turnover time is not universal and dependent on these winds. The models yield results in good agreement with direct numerical simulations of stratified turbulence, for which single-particle dispersion differs from the well-studied case of homogeneous and isotropic turbulence.

Optimal energy growth in a stably stratified shear flow

NASA Astrophysics Data System (ADS)

Jose, Sharath; Roy, Anubhab; Bale, Rahul; Iyer, Krithika; Govindarajan, Rama

2018-02-01

Transient growth of perturbations by a linear non-modal evolution is studied here in a stably stratified bounded Couette flow. The density stratification is linear. Classical inviscid stability theory states that a parallel shear flow is stable to exponentially growing disturbances if the Richardson number (Ri) is greater than 1/4 everywhere in the flow. Experiments and numerical simulations at higher Ri show however that algebraically growing disturbances can lead to transient amplification. The complexity of a stably stratified shear flow stems from its ability to combine this transient amplification with propagating internal gravity waves (IGWs). The optimal perturbations associated with maximum energy amplification are numerically obtained at intermediate Reynolds numbers. It is shown that in this wall-bounded flow, the three-dimensional optimal perturbations are oblique, unlike in unstratified flow. A partitioning of energy into kinetic and potential helps in understanding the exchange of energies and how it modifies the transient growth. We show that the apportionment between potential and kinetic energy depends, in an interesting manner, on the Richardson number, and on time, as the transient growth proceeds from an optimal perturbation. The oft-quoted stabilizing role of stratification is also probed in the non-diffusive limit in the context of disturbance energy amplification.

Photon Counting System for High-Sensitivity Detection of Bioluminescence at Optical Fiber End.

PubMed

Iinuma, Masataka; Kadoya, Yutaka; Kuroda, Akio

2016-01-01

The technique of photon counting is widely used for various fields and also applicable to a high-sensitivity detection of luminescence. Thanks to recent development of single photon detectors with avalanche photodiodes (APDs), the photon counting system with an optical fiber has become powerful for a detection of bioluminescence at an optical fiber end, because it allows us to fully use the merits of compactness, simple operation, highly quantum efficiency of the APD detectors. This optical fiber-based system also has a possibility of improving the sensitivity to a local detection of Adenosine triphosphate (ATP) by high-sensitivity detection of the bioluminescence. In this chapter, we are introducing a basic concept of the optical fiber-based system and explaining how to construct and use this system.

Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells.

PubMed

Limon-Boulez, I; Bouet-Alard, R; Gettys, T W; Lanier, S M; Maltier, J P; Legrand, C

2001-02-01

alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR > alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

The first report of luminescent liver tissue in fishes: evolution and structure of bioluminescent organs in the deep-sea naked barracudinas (Aulopiformes: Lestidiidae).

PubMed

Ghedotti, Michael J; Barton, Ryan W; Simons, Andrew M; Davis, Matthew P

2015-03-01

Bioluminescent organs that provide ventral camouflage are common among fishes in the meso-bathypelagic zones of the deep sea. However, the anatomical structures that have been modified to produce light vary substantially among different groups of fishes. Although the anatomical structure and evolutionary derivation of some of these organs have been well studied, the light organs of the naked barracudinas have received little scientific attention. This study describes the anatomy and evolution of bioluminescent organs in the Lestidiidae (naked barracudinas) in the context of a new phylogeny of barracudinas and closely related alepisauroid fishes. Gross and histological examination of bioluminescent organs or homologous structures from preserved museum specimens indicate that the ventral light organ is derived from hepatopancreatic tissue and that the antorbital spot in Lestrolepis is, in fact, a second dermal light organ. In the context of the phylogeny generated from DNA-sequence data from eight gene fragments (7 nuclear and 1 mitochondrial), a complex liver with a narrow ventral strand running along the ventral midline evolves first in the Lestidiidae. The ventral hepatopancreatic tissue later evolves into a ventral bioluminescent organ in the ancestor of Lestidium and Lestrolepis with the lineage leading to the genus Lestrolepis evolving a dermal antorbital bioluminescent organ, likely for light-intensity matching. This is the first described hepatopancreatic bioluminescent organ in fishes. © 2014 Wiley Periodicals, Inc.

Crystal structure of native and a mutant of Lampyris turkestanicus luciferase implicate in bioluminescence color shift.

PubMed

Kheirabadi, Mitra; Sharafian, Zohreh; Naderi-Manesh, Hossein; Heineman, Udo; Gohlke, Ulrich; Hosseinkhani, Saman

2013-12-01

Firefly bioluminescence reaction in the presence of Mg(2+), ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350-359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7Å and 2.2Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351-359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission. © 2013.

In vivo quantitative bioluminescence tomography using heterogeneous and homogeneous mouse models.

PubMed

Liu, Junting; Wang, Yabin; Qu, Xiaochao; Li, Xiangsi; Ma, Xiaopeng; Han, Runqiang; Hu, Zhenhua; Chen, Xueli; Sun, Dongdong; Zhang, Rongqing; Chen, Duofang; Chen, Dan; Chen, Xiaoyuan; Liang, Jimin; Cao, Feng; Tian, Jie

2010-06-07

Bioluminescence tomography (BLT) is a new optical molecular imaging modality, which can monitor both physiological and pathological processes by using bioluminescent light-emitting probes in small living animal. Especially, this technology possesses great potential in drug development, early detection, and therapy monitoring in preclinical settings. In the present study, we developed a dual modality BLT prototype system with Micro-computed tomography (MicroCT) registration approach, and improved the quantitative reconstruction algorithm based on adaptive hp finite element method (hp-FEM). Detailed comparisons of source reconstruction between the heterogeneous and homogeneous mouse models were performed. The models include mice with implanted luminescence source and tumor-bearing mice with firefly luciferase report gene. Our data suggest that the reconstruction based on heterogeneous mouse model is more accurate in localization and quantification than the homogeneous mouse model with appropriate optical parameters and that BLT allows super-early tumor detection in vivo based on tomographic reconstruction of heterogeneous mouse model signal.

Transfection in perfused microfluidic cell culture devices: A case study.

PubMed

Raimes, William; Rubi, Mathieu; Super, Alexandre; Marques, Marco P C; Veraitch, Farlan; Szita, Nicolas

2017-08-01

Automated microfluidic devices are a promising route towards a point-of-care autologous cell therapy. The initial steps of induced pluripotent stem cell (iPSC) derivation involve transfection and long term cell culture. Integration of these steps would help reduce the cost and footprint of micro-scale devices with applications in cell reprogramming or gene correction. Current examples of transfection integration focus on maximising efficiency rather than viable long-term culture. Here we look for whole process compatibility by integrating automated transfection with a perfused microfluidic device designed for homogeneous culture conditions. The injection process was characterised using fluorescein to establish a LabVIEW-based routine for user-defined automation. Proof-of-concept is demonstrated by chemically transfecting a GFP plasmid into mouse embryonic stem cells (mESCs). Cells transfected in the device showed an improvement in efficiency (34%, n = 3) compared with standard protocols (17.2%, n = 3). This represents a first step towards microfluidic processing systems for cell reprogramming or gene therapy.

Bioluminescence Imaging of Clavibacter michiganensis subsp. michiganensis Infection of Tomato Seeds and Plants ▿

PubMed Central

Xu, Xiulan; Miller, Sally A.; Baysal-Gurel, Fulya; Gartemann, Karl-Heinz; Eichenlaub, Rudolf; Rajashekara, Gireesh

2010-01-01

Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the

Effects of molecular size and chemical factor on plasma gene transfection

NASA Astrophysics Data System (ADS)

Ikeda, Yoshihisa; Motomura, Hideki; Kido, Yugo; Satoh, Susumu; Jinno, Masafumi

2016-07-01

In order to clarify the mechanism of plasma gene transfection, the relationship between transfection efficiency and transferred molecular size was investigated. Molecules with low molecular mass (less than 50 kDa; dye or dye-labeled oligonucleotide) and high molecular mass (more than 1 MDa; plasmid DNA or fragment of plasmid DNA) were transferred to L-929 cells. It was found that the transfection efficiency decreases with increasing in transferred molecular size and also depends on the tertiary structure of transferred molecules. Moreover, it was suggested the transfection mechanism is different between the molecules with low (less than 50 kDa) and high molecular mass (higher than 1 MDa). For the amount of gene transfection after plasma irradiation, which is comparable to that during plasma irradiation, it is shown that H2O2 molecules are the main contributor. The transfection efficiency decreased to 0.40 ± 0.22 upon scavenging the H2O2 generated by plasma irradiation using the catalase. On the other hand, when the H2O2 solution is dropped into the cell suspension without plasma irradiation, the transfection efficiency is almost 0%. In these results, it is also suggested that there is a synergetic effect of H2O2 with electrical factors or other reactive species generated by plasma irradiation.

Application of the freeze-dried bioluminescent bioreporter Pseudomonas putida mt-2 KG1206 to the biomonitoring of groundwater samples from monitoring wells near gasoline leakage sites.

PubMed

Ko, Kyung-Seok; Kong, In Chul

2017-02-01

This study examined the applicability of a freeze-dried bioluminescent bioreporter, Pseudomonas putida mt-2 KG1206 (called KG1206), to the biomonitoring of groundwater samples. Samples were collected from the monitoring wells of gas station tanks or old pipeline leakage sites in Korea. In general, the freeze-dried strain in the presence of pure inducer chemicals showed low bioluminescence activity and a different activity order compared with that of the subcultured strain. The effects of KNO 3 as a bioluminescence stimulant were observed on the pure inducers and groundwater samples. The stimulation rates varied according to the type of inducers and samples, ranging from 2.2 to 20.5 times (for pure inducers) and from 1.1 to 11 times (for groundwater samples) the total bioluminescence of the control. No considerable correlations were observed between the bioluminescence intensity of the freeze-dried strain and the inducer concentrations in the samples (R 2  < 0.1344). However, samples without a high methyl tertiary butyl ether (MTBE) level and those from the gas station leakage site showed reasonable correlations with the bioluminescence activity with R 2 values of 0.3551 and 0.4131, respectively. These results highlight the potential of using freeze-dried bioluminescent bacteria as a rapid, simple, and portable tool for the preliminary biomonitoring of specific pollutants at contaminated sites.

BarTeL, a Genetically Versatile, Bioluminescent and Granule Neuron Precursor-Targeted Mouse Model for Medulloblastoma

PubMed Central

Mahdi, Min Y.; Gonzalez-Gomez, Ignacio; Asgharzadeh, Shahab; D’Apuzzo, Massimo; Erdreich-Epstein, Anat; Moats, Rex A.

2016-01-01

Medulloblastomas are the most common malignant pediatric brain tumor and have been divided into four major molecular subgroups. Animal models that mimic the principal molecular aberrations of these subgroups will be important tools for preclinical studies and allow greater understanding of medulloblastoma biology. We report a new transgenic model of medulloblastoma that possesses a unique combination of desirable characteristics including, among others, the ability to incorporate multiple and variable genes of choice and to produce bioluminescent tumors from a limited number of somatic cells within a normal cellular environment. This model, termed BarTeL, utilizes a Barhl1 homeobox gene promoter to target expression of a bicistronic transgene encoding both the avian retroviral receptor TVA and an eGFP-Luciferase fusion protein to neonatal cerebellar granule neuron precursor (cGNP) cells, which are cells of origin for the sonic hedgehog (SHH) subgroup of human medulloblastomas. The Barhl1 promoter-driven transgene is expressed strongly in mammalian cGNPs and weakly or not at all in mature granule neurons. We efficiently induced bioluminescent medulloblastomas expressing eGFP-luciferase in BarTeL mice by infection of a limited number of somatic cGNPs with avian retroviral vectors encoding the active N-terminal fragment of SHH and a stabilized MYCN mutant. Detection and quantification of the increasing bioluminescence of growing tumors in young BarTeL mice was facilitated by the declining bioluminescence of their uninfected maturing cGNPs. Inclusion of eGFP in the transgene allowed enriched sorting of cGNPs from neonatal cerebella. Use of a single bicistronic avian vector simultaneously expressing both Shh and Mycn oncogenes increased the medulloblastoma incidence and aggressiveness compared to mixed virus infections. Bioluminescent tumors could also be produced by ex vivo transduction of neonatal BarTeL cerebellar cells by avian retroviruses and subsequent

Transient transfection of intraerythrocytic Babesia gibsoni using elongation factor-1 alpha promoter.

PubMed

Liu, Mingming; Asada, Masahito; Cao, Shinuo; Adjou Moumouni, Paul Franck; Vudriko, Patrick; Efstratiou, Artemis; Hakimi, Hassan; Masatani, Tatsunori; Sunaga, Fujiko; Kawazu, Shin-Ichiro; Yamagishi, Junya; Xuan, Xuenan

2017-09-01

The development of gene manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not yet been established for Babesia gibsoni. Here, we report for the first time, the successful transient transfection of B. gibsoni. The plasmid containing the firefly luciferase reporter gene (pBS-ELA) was transfected into B. gibsoni by an AMAXA 4D Nucleofector™ device. Transfection using program FA113 and Lonza buffer SF showed the highest luciferase expression. Twenty micrograms of plasmid produced the highest relative transfection efficiency. The fluorescent protein-expressing parasites were determined by GFP-containing plasmid (pBS-EGA) at 48 and 72h post transfection. This finding is the first step towards a stable transfection method for B. gibsoni, which may contribute to a better understanding of the biology of the parasite. Copyright © 2017 Elsevier B.V. All rights reserved.

Toward Contactless Biology: Acoustophoretic DNA Transfection

NASA Astrophysics Data System (ADS)

Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

2016-02-01

Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

Toward Contactless Biology: Acoustophoretic DNA Transfection.

PubMed

Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

2016-02-01

Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors.

Toward Contactless Biology: Acoustophoretic DNA Transfection

PubMed Central

Vasileiou, Thomas; Foresti, Daniele; Bayram, Adem; Poulikakos, Dimos; Ferrari, Aldo

2016-01-01

Acoustophoresis revolutionized the field of container-less manipulation of liquids and solids by enabling mixing procedures which avoid contamination and loss of reagents due to the contact with the support. While its applications to chemistry and engineering are straightforward, additional developments are needed to obtain reliable biological protocols in a contactless environment. Here, we provide a first, fundamental step towards biological reactions in air by demonstrating the acoustophoretic DNA transfection of mammalian cells. We developed an original acoustophoretic design capable of levitating, moving and mixing biological suspensions of living mammalians cells and of DNA plasmids. The precise and sequential delivery of the mixed solutions into tissue culture plates is actuated by a novel mechanism based on the controlled actuation of the acoustophoretic force. The viability of the contactless procedure is tested using a cellular model sensitive to small perturbation of neuronal differentiation pathways. Additionally, the efficiency of the transfection procedure is compared to standard, container-based methods for both single and double DNA transfection and for different cell types including adherent growing HeLa cancer cells, and low adhesion neuron-like PC12 cells. In all, this work provides a proof of principle which paves the way to the development of high-throughput acoustophoretic biological reactors. PMID:26828312

Mechanism of energy conversion and transfer in bioluminescence. Final report. [Sea pansy Renilla reniformis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cormier, M.J.

1979-01-01

Bioluminescence in the sea pansy, Renilla reniformis, a marine anthozoan coelenterate, is a complex process involving the participation of three proteins specific to anthozoan coelenterate-type systems. These are: (1) the luciferin binding protein, (2) the enzyme luciferase, and (3) the green-fluorescent protein. Each of these have been purified and characterized and the structure of luciferin has been confirmed by synthesis. Luciferin binding protein (BP-LH/sub 2/) is a specific substrate binding protein which binds one molecule of coelenterate-type luciferin per molecule of protein and which then releases luciferin in the presence of Ca/sup + +/. Luciferase is the enzyme which catalyzesmore » oxidation (by O/sub 2/) of coelenterate-type luciferin, leading to the production of CO/sub 2/ and enzyme-bound, excited-state oxyluciferin. Oxyluciferin may then emit blue light by a direct de-excitation pathway or may transfer excitation energy to the green-fluorescent protein (GFP). GFP is a non-catalytic accessory protein which accepts excitation energy from oxyluciferin, by radiationless energy transfer, and then emits green bioluminescence. The Renilla bioluminescence system is thus the first radiationless energy transfer system the individual components of which have been purified to homogeneity, characterized, and then reassembled in vitro with restoration of the energy transfer function.« less

Impact of Site-Directed Mutant Luciferase on Quantitative Green and Orange/Red Emission Intensities in Firefly Bioluminescence

NASA Astrophysics Data System (ADS)

Wang, Yu; Akiyama, Hidefumi; Terakado, Kanako; Nakatsu, Toru

2013-08-01

Firefly bioluminescence has attracted great interest because of its high quantum yield and intriguing modifiable colours. Modifications to the structure of the enzyme luciferase can change the emission colour of firefly bioluminescence, and the mechanism of the colour change has been intensively studied by biochemists, structural biologists, optical physicists, and quantum-chemistry theorists. Here, we report on the quantitative spectra of firefly bioluminescence catalysed by wild-type and four site-directed mutant luciferases. While the mutation caused different emission spectra, the spectra differed only in the intensity of the green component (λmax ~ 560 nm). In contrast, the orange (λmax ~ 610 nm) and red (λmax ~ 650 nm) components present in all the spectra were almost unaffected by the modifications to the luciferases and changes in pH. Our results reveal that the intensity of the green component is the unique factor that is influenced by the luciferase structure and other reaction conditions.

Hydrophobic modification of low molecular weight polyethylenimine for improved gene transfection.

PubMed

Teo, Pei Yun; Yang, Chuan; Hedrick, James L; Engler, Amanda C; Coady, Daniel J; Ghaem-Maghami, Sadaf; George, Andrew J T; Yang, Yi Yan

2013-10-01

Hydrophobic modification of low molecular weight (LMW) polyethylenimine (PEI) is known to increase gene transfection efficiency of LMW PEI. However, few studies have explored how the conjugated hydrophobic groups influence the properties of the modified LMW PEI mainly due to difficulties in obtaining well defined final product compositions and limitations in current chemical synthesis routes. The aim of this study was to modify LMW PEI (Mn 1.8 kDa, PEI-1.8) judiciously with different hydrophobic functional groups and to investigate how hydrophobicity, molecular structure and inclusion of hydrogen bonding properties in the conjugated side groups as well as the conjugation degree (number of primary amine groups of PEI-1.8 modified with hydrophobic groups) influence PEI-1.8 gene transfection efficiency. The modified polymers were characterized for DNA binding ability, particle size, zeta potential, in vitro gene transfection efficiency and cytotoxicity in SKOV-3 human ovarian cancer and HepG2 human liver carcinoma cell lines. The study shows that modified PEI-1.8 polymers are able to condense plasmid DNA into cationic nanoparticles, of sizes ~100 nm, whereas unmodified polymer/DNA complexes display larger particle sizes of 2 μm. Hydrophobic modification also increases the zeta potential of polymer/DNA complexes. Importantly, modified PEI-1.8 shows enhanced transfection efficiency over the unmodified counterpart. Higher transfection efficiency is obtained when PEI-1.8 is modified with shorter hydrophobic groups (MTC-ethyl) as opposed to longer ones (MTC-octyl and MTC-deodecyl). An aromatic structured functional group (MTC-benzyl) also enhances transfection efficiency more than an alkyl functional group (MTC-octyl). An added hydrogen-bonding urea group in the conjugated functional group (MTC-urea) does not enhance transfection efficiency over one without urea (MTC-benzyl). The study also demonstrates that modification degree greatly influences gene transfection, and

Ultrasound modulation of bioluminescence generated inside a turbid medium

NASA Astrophysics Data System (ADS)

Ahmad, Junaid; Jayet, Baptiste; Hill, Philip J.; Mather, Melissa L.; Dehghani, Hamid; Morgan, Stephen P.

2017-03-01

In vivo bioluminescence imaging (BLI) has poor spatial resolution owing to strong light scattering by tissue, which also affects quantitative accuracy. This paper proposes a hybrid acousto-optic imaging platform that images bioluminescence modulated at ultrasound (US) frequency inside an optically scattering medium. This produces an US modulated light within the tissue that reduces the effects of light scattering and improves the spatial resolution. The system consists of a continuously excited 3.5 MHz US transducer applied to a tissue like phantom of known optical properties embedded with bio-or chemiluminescent sources that are used to mimic in vivo experiments. Scanning US over the turbid medium modulates the luminescent sources deep inside tissue at several US scan points. These modulated signals are recorded by a photomultiplier tube and lock-in detection to generate a 1D profile. Indeed, high frequency US enables small focal volume to improve spatial resolution, but this leads to lower signal-to-noise ratio. First experimental results show that US enables localization of a small luminescent source (around 2 mm wide) deep ( 20 mm) inside a tissue phantom having a scattering coefficient of 80 cm-1. Two sources separated by 10 mm could be resolved 20 mm inside a chicken breast.

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

PubMed Central

Felgner, P L; Gadek, T R; Holm, M; Roman, R; Chan, H W; Wenz, M; Northrop, J P; Ringold, G M; Danielsen, M

1987-01-01

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique. Images PMID:2823261

Novel Bioluminescent Activatable Reporter for Src Tyrosine Kinase Activity in Living Mice

PubMed Central

Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng

2016-01-01

Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850

Noninvasive bioluminescence imaging of normal and spontaneously transformed prostate tissue in mice.

PubMed

Lyons, Scott K; Lim, Ed; Clermont, Anne O; Dusich, Joan; Zhu, Lingyun; Campbell, Kenneth D; Coffee, Richard J; Grass, David S; Hunter, John; Purchio, Tony; Jenkins, Darlene

2006-05-01

Several transgenic mouse models of prostate cancer have been developed recently that are able to recapitulate many key biological features of the human condition. It would, therefore, be desirable to employ these models to test the efficacy of new therapeutics before clinical trial; however, the variable onset and non-visible nature of prostate tumor development limit their use for such applications. We now report the generation of a transgenic reporter mouse that should obviate these limitations by enabling noninvasive in vivo bioluminescence imaging of normal and spontaneously transformed prostate tissue in the mouse. We used an 11-kb fragment of the human prostate-specific antigen (PSA) promoter to achieve specific and robust expression of firefly luciferase in the prostate glands of transgenic mice. Ex vivo bioluminescence imaging and in situ hybridization analysis confirmed that luciferase expression was restricted to the epithelium in all four lobes of the prostate. We also show that PSA-Luc mice exhibit decreased but readily detectable levels of in vivo bioluminescence over extended time periods following androgen ablation. These results suggest that this reporter should enable in vivo imaging of both androgen-dependent and androgen-independent prostate tumor models. As proof-of-principle, we show that we could noninvasively image SV40 T antigen-induced prostate tumorigenesis in mice with PSA-Luc. Furthermore, we show that our noninvasive imaging strategy can be successfully used to image tumor response to androgen ablation in transgenic mice and, as a result, that we can rapidly identify individual animals capable of sustaining tumor growth in the absence of androgen.

Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamaguchi, Kazuki; Department of Anatomy, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City 814-0180; Feril, Loreto B., E-mail: ferilism@yahoo.com

2011-07-22

Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA,more » which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.« less

Alkaline polymer electrolyte fuel cells stably working at 80 °C

NASA Astrophysics Data System (ADS)

Peng, Hanqing; Li, Qihao; Hu, Meixue; Xiao, Li; Lu, Juntao; Zhuang, Lin

2018-06-01

Alkaline polymer electrolyte fuel cells are a new class of polymer electrolyte fuel cells that fundamentally enables the use of nonprecious metal catalysts. The cell performance mostly relies on the quality of alkaline polymer electrolytes, including the ionic conductivity and the chemical/mechanical stability. For a long time, alkaline polymer electrolytes are thought to be too weak in stability to allow the fuel cell to be operated at elevated temperatures, e.g., above 60 °C. In the present work, we report a progress in the state-of-the-art alkaline polymer electrolyte fuel cell technology. By using a newly developed alkaline polymer electrolyte, quaternary ammonia poly (N-methyl-piperidine-co-p-terphenyl), which simultaneously possesses high ionic conductivity and excellent chemical/mechanical stability, the fuel cell can now be stably operated at 80 °C with high power density. The peak power density reaches ca. 1.5 W/cm2 at 80 °C with Pt/C catalysts used in both the anode and the cathode. The cell works stably in a period of study over 100 h.

Observations and Measurements of Planktonic Bioluminescence in and Around a Milky Sea

DTIC Science & Technology

1988-03-01

malticharnel analysers operating in the multiscaler mode. The details of both the onboard underway system and the LPTC systems have been published (Lapota...the Arabian Sea during the southwest monsoon. No nutrient data was collected during our study, yet phosphates, nitrates , and trace BIOLUMINESCENCE IN

Enhanced transfection by antioxidative polymeric gene carrier that reduces polyplex-mediated cellular oxidative stress.

PubMed

Lee, Min Sang; Kim, Nak Won; Lee, Kyuri; Kim, Hongtae; Jeong, Ji Hoon

2013-06-01

To test the hypothesis in which polyplex-induced oxidative stress may affect overall transfection efficiency, an antioxidative transfection system minimizing cellular oxidative stress was designed for enhanced transfection. An amphiphilic copolymer (PEI-PLGA) was synthesized and used as a micelle-type gene carrier containing hydrophobic antioxidant, α-tocopherol. Cellular oxidative stress and the change of mitochondrial membrane potential after transfection was measured by using a fluorescent probe (H₂DCFDA) and lipophilic cationic probe (JC-1), respectively. Transfection efficiency was determined by measuring a reporter gene (luciferase) expression level. The initial transfection study with conventional PEI/plasmid DNA polyplex showed significant generation of reactive oxygen species (ROS). The PEI-PLGA copolymer successfully carried out the simultaneous delivery of α-tocopherol and plasmid DNA (PEI-PLGA/Toco/pDNA polyplex) into cells, resulting in a significant reduction in cellular ROS generation after transfection and helped to maintain the mitochondrial membrane potential (ΔΨ). In addition, the transfection efficiency was dramatically increased using the antioxidative transfection system. This work showed that oxidative stress would be one of the important factors that should be considered in designing non-viral gene carriers and suggested a possible way to reduce the carrier-mediated oxidative stress, which consequently leads to enhanced transfection.

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

PubMed

Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

2015-07-15

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity

PubMed Central

2015-01-01

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain. PMID:26120870

Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca2+ microscopy.

PubMed

Suzuki, Kazushi; Onishi, Takahito; Nakada, Chieko; Takei, Shunsuke; Daniels, Matthew J; Nakano, Masahiro; Matsuda, Tomoki; Nagai, Takeharu

2018-05-18

Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for high-throughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca 2+ indicator GmNL(Ca 2+ ), and its application in a customized microscope for high-throughput drug screening. GmNL(Ca 2+ ) gives a 140% signal change with Ca 2+ , and can image drug-induced changes of Ca 2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca 2+ ) with this adaptation, we could image spontaneous Ca 2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.

The bioluminescent Listeria monocytogenes strain Xen32 is defective in flagella expression and highly attenuated in orally infected BALB/cJ mice.

PubMed

Bergmann, Silke; Rohde, Manfred; Schughart, Klaus; Lengeling, Andreas

2013-07-15

In vivo bioluminescence imaging (BLI) is a powerful method for the analysis of host-pathogen interactions in small animal models. The commercially available bioluminescent Listeria monocytogenes strain Xen32 is commonly used to analyse immune functions in knockout mice and pathomechanisms of listeriosis. To analyse and image listerial dissemination after oral infection we have generated a murinised Xen32 strain (Xen32-mur) which expresses a previously described mouse-adapted internalin A. This strain was used alongside the Xen32 wild type strain and the bioluminescent L. monocytogenes strains EGDe-lux and murinised EGDe-mur-lux to characterise bacterial dissemination in orally inoculated BALB/cJ mice. After four days of infection, Xen32 and Xen32-mur infected mice displayed consistently higher rates of bioluminescence compared to EGDe-lux and EGDe-mur-lux infected animals. However, surprisingly both Xen32 strains showed attenuated virulence in orally infected BALB/c mice that correlated with lower bacterial burden in internal organs at day 5 post infection, smaller losses in body weights and increased survival compared to EGDe-lux or EGDe-mur-lux inoculated animals. The Xen32 strain was made bioluminescent by integration of a lux-kan transposon cassette into the listerial flaA locus. We show here that this integration results in Xen32 in a flaA frameshift mutation which makes this strain flagella deficient. The bioluminescent L. monocytogenes strain Xen32 is deficient in flagella expression and highly attenuated in orally infected BALB/c mice. As this listerial strain has been used in many BLI studies of murine listeriosis, it is important that the scientific community is aware of its reduced virulence in vivo.

Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

PubMed Central

Gandelman, Olga A.; Church, Vicki L.; Moore, Cathy A.; Kiddle, Guy; Carne, Christopher A.; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C.; Murray, James A. H.

2010-01-01

Background The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Principal Findings Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. Conclusions The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light

[Detection of stable expression of human interlukin-2 gene in transfected keratinocytes].

PubMed

Liao, W; Liu, Y; Ye, L

1999-09-01

To investigate the stable expression and secretion of human interlukin-2 gene in transfected keratinocytes. Keratinocytes were transfected with lipofectamine and selected by G418. Then the samples were analyzed with the techniques of DNA dot blot, RNA dot blot, hybridization in situ, immunohistochemistry, Western blot and MTT. The positive signals were observed in transfected keratinocytes by DNA dot blot, RNA dot blot, hybridization in situ and immunohistochemistry. With Western blot analysis, a specific band exhibiting a molecular weight of 15,000 was detected in transfected keratinocytes, which was in acordance with that of IL-2. The expression of IL-2 can maintain for up to 1 month. The amounts of IL-2 in the supernatants of two and four passages transfected keratinocytes were 27.7 U/ml and 15.0 U/ml, respectively. Keratinocytes have the potential for stable gene expression and secretion of active transgene products. Thus, it is possible to use keratinocytes as a target cell for gene transfection, gene expression and even gene therapy.

Superhydrophilic-Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection.

PubMed

Ueda, Erica; Feng, Wenqian; Levkin, Pavel A

2016-10-01

High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal transfection reagent, ScreenFect A, the method of reverse cell transfection is optimized on the patterned substrates and several factors that affect transfection efficiency and cytotoxicity are identified. Higher levels of transfection are achieved on HOOC- versus NH 2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the transfection mixture, while minimizing the amount of transfection reagent improves cell viability. Almost no diffusion of the printed transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing reverse cell transfection conditions before performing further cell screenings. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations.

PubMed

Amaral, Danilo T; Oliveira, Gabriela; Silva, Jaqueline R; Viviani, Vadim R

2016-08-31

Bioluminescent click-beetles display a wide variation of bioluminescence colors ranging from green to orange, including an unusual intra-specific color variation in the Jamaican Pyrophorus plagiophthalamus. Recently, we collected individuals of the Pyrophorus angustus species from the Southern Amazon forest, in Brazil, which displays an orange light emitting abdominal lantern. This species was also previously described from Central America, but displaying a bioluminescence spectrum from 536 nm (dorsal) to 578 nm (ventral). The biogeographic variation of the bioluminescence color in this species could be an adaptation to environmental reflectance and inter/intraspecific sexual competition. Here, we cloned, sequenced, characterized and performed site-direct mutagenesis of this new orange emitting luciferase. The in vitro luciferase spectrum displayed a peak at 594 nm, KM values for ATP and d-luciferin of 160 μM and 17 μM, respectively, and an optimum pH of approximately 8.5. Comparative multialignment and site-directed mutagenesis using different color emitting click-beetle luciferases from P. angustus, Fulgeochlizus bruchi and Pyrearinus termitilluminans luciferases cloned by our group showed an integral role of residue 247 in bioluminescence color modulation.

Prolonged bioluminescence imaging in living cells and mice using novel pro-substrates for Renilla luciferase.

PubMed

Yuan, Mingliang; Ma, Xiaojie; Jiang, Tianyu; Gao, Yuqi; Cui, Yuanyuan; Zhang, Chaochao; Yang, Xingye; Huang, Yun; Du, Lupei; Yampolsky, Ilia; Li, Minyong

2017-12-13

The prodrug or caged-luciferin strategy affords an excellent platform for persistent bioluminescence imaging. In the current work, we designed and synthesized ten novel pro-substrates for Renilla luciferase by introducing ester protecting groups of different sizes into the carbonyl group of the free luciferin 1. Taking advantage of intracellular esterases, lipases, and nucleophilic substances, the ester protecting groups were hydrolyzed, resulting in the release of a free luciferin and a bioluminescence signal turn-on. Among the tested pro-substrates, the butyryloxymethyl luciferin 7 exhibited low cytotoxicity and a prolonged luminescence signal both in cellulo and in vivo. Therefore, the butyryloxymethyl luciferin 7 can act as a promising substrate for noninvasive extended imaging in diagnostic and therapeutic fields.

Photoenhanced gene transfection by a curcumin loaded CS-g-PZLL micelle.

PubMed

Lin, Jian-Tao; Pan, Wen-Jia; Zhang, Jun-Ai; Wang, Wei; Zhong, Jia; Su, Jia-Min; Li, Tong; Zou, Ying; Wang, Guan-Hai

2017-09-01

The codelivery of drug and gene is a promising method for cancer treatment. In our previous works, we prepared a cationic micelles based on chitosan and poly-(N-3-carbobenzyloxylysine) (CS-g-PZLL), but transfection ratio of CS-g-PZLL to Hela cell was low. Herein, to improve the transfection efficiency of CS-g-PZLL, curcumin was loaded in the CS-g-PZLL micelle. After irradiation, the obtained curcumin loaded micelle showed a better transfection, and the p53 protein expression in Hela cells was higher. The apoptosis assay showed that the complex could induce a more significant apoptosis to Hela cells than that of curcumin or p53 used alone, and the curcumin loaded micelle inducing apoptosis was best after irradiation. Therefore, CS-g-PZLL is a safe and effective carrier for the codelivery of drug/gene, and curcumin could be used as a photosensitizer to induce a photoenhanced gene transfection, which should be encouraged in improving transfection and tumor therapy. Copyright © 2017. Published by Elsevier B.V.

Synthesis of linear polyethylenimine derivatives for DNA transfection.

PubMed

Brissault, Blandine; Kichler, Antoine; Guis, Christine; Leborgne, Christian; Danos, Olivier; Cheradame, Hervé

2003-01-01

A series of linear polymers containing varying amounts of ethylenimine or N-propylethylenimine units were synthesized by hydrolysis and/or reduction of polyethyloxazolines. The pK(a)s of the polyamines were determined potentiometrically. Gel mobility shift assay showed that the efficiency of DNA complexation was related to the fraction of amino groups that are protonated at neutral pH. The effects of cationic charge density and molar weight of the polymers on the transfection efficiency were evaluated on HepG2 cells. The results obtained with different copolymers show that the transfection efficiency primarily depends on the fraction of ethylenimine units included in the polymer albeit the molar weight is also of importance. On the basis of the results obtained with poly(N-propylethylenimines), we also demonstrate that the high transfection efficiency of polyethylenimines does not solely rely on their capacity to capture protons which are transferred into the endo-lysosomes during acidification.

Brazilian Bioluminescent Beetles: Reflections on Catching Glimpses of Light in the Atlantic Forest and Cerrado.

PubMed

Bechara, Etelvino J H; Stevani, Cassius V

2018-01-01

Bioluminescence - visible and cold light emission by living organisms - is a worldwide phenomenon, reported in terrestrial and marine environments since ancient times. Light emission from microorganisms, fungi, plants and animals may have arisen as an evolutionary response against oxygen toxicity and was appropriated for sexual attraction, predation, aposematism, and camouflage. Light emission results from the oxidation of a substrate, luciferin, by molecular oxygen, catalyzed by a luciferase, producing oxyluciferin in the excited singlet state, which decays to the ground state by fluorescence emission. Brazilian Atlantic forests and Cerrados are rich in luminescent beetles, which produce the same luciferin but slightly mutated luciferases, which result in distinct color emissions from green to red depending on the species. This review focuses on chemical and biological aspects of Brazilian luminescent beetles (Coleoptera) belonging to the Lampyridae (fireflies), Elateridae (click-beetles), and Phengodidae (railroad-worms) families. The ATP-dependent mechanism of bioluminescence, the role of luciferase tuning the color of light emission, the "luminous termite mounds" in Central Brazil, the cooperative roles of luciferase and superoxide dismutase against oxygen toxicity, and the hypothesis on the evolutionary origin of luciferases are highlighted. Finally, we point out analytical uses of beetle bioluminescence for biological, clinical, environmental, and industrial samples.

Improving image reconstruction of bioluminescence imaging using a priori information from ultrasound imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Jayet, Baptiste; Ahmad, Junaid; Taylor, Shelley L.; Hill, Philip J.; Dehghani, Hamid; Morgan, Stephen P.

2017-03-01

Bioluminescence imaging (BLI) is a commonly used imaging modality in biology to study cancer in vivo in small animals. Images are generated using a camera to map the optical fluence emerging from the studied animal, then a numerical reconstruction algorithm is used to locate the sources and estimate their sizes. However, due to the strong light scattering properties of biological tissues, the resolution is very limited (around a few millimetres). Therefore obtaining accurate information about the pathology is complicated. We propose a combined ultrasound/optics approach to improve accuracy of these techniques. In addition to the BLI data, an ultrasound probe driven by a scanner is used for two main objectives. First, to obtain a pure acoustic image, which provides structural information of the sample. And second, to alter the light emission by the bioluminescent sources embedded inside the sample, which is monitored using a high speed optical detector (e.g. photomultiplier tube). We will show that this last measurement, used in conjunction with the ultrasound data, can provide accurate localisation of the bioluminescent sources. This can be used as a priori information by the numerical reconstruction algorithm, greatly increasing the accuracy of the BLI image reconstruction as compared to the image generated using only BLI data.

Use of a highly sensitive two-dimensional luminescence imaging system to monitor endogenous bioluminescence in plant leaves

PubMed Central

Flor-Henry, Michel; McCabe, Tulene C; de Bruxelles, Guy L; Roberts, Michael R

2004-01-01

Background All living organisms emit spontaneous low-level bioluminescence, which can be increased in response to stress. Methods for imaging this ultra-weak luminescence have previously been limited by the sensitivity of the detection systems used. Results We developed a novel configuration of a cooled charge-coupled device (CCD) for 2-dimensional imaging of light emission from biological material. In this study, we imaged photon emission from plant leaves. The equipment allowed short integration times for image acquisition, providing high resolution spatial and temporal information on bioluminescence. We were able to carry out time course imaging of both delayed chlorophyll fluorescence from whole leaves, and of low level wound-induced luminescence that we showed to be localised to sites of tissue damage. We found that wound-induced luminescence was chlorophyll-dependent and was enhanced at higher temperatures. Conclusions The data gathered on plant bioluminescence illustrate that the equipment described here represents an improvement in 2-dimensional luminescence imaging technology. Using this system, we identify chlorophyll as the origin of wound-induced luminescence from leaves. PMID:15550176

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR).

PubMed

Crosby, Priya; Hoyle, Nathaniel P; O'Neill, John S

2017-12-13

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Prolonging the expression duration of ultrasound-mediated gene transfection using PEI nanoparticles.

PubMed

Lee, Jyun-Lin; Lo, Chia-Wen; Ka, Shuk-Man; Chen, Ann; Chen, Wen-Shiang

2012-05-30

Ultrasound (US) irradiation has been found to facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, its transfection efficiency is generally low, and the expression duration of transfected gene is short. Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. The purpose of this study is to determine whether PEI can also prolong the expression duration after US-mediated transfection. A mixture of pCMViLUC and 22-kDa linear PEI was transfected to cultured cells or mouse muscle by exposure to 1-MHz pulsed US. The duration of expression was assessed periodically following US treatment. As expected, strong expression of luciferase could be found 30days after the treatment of DNA-PEI complex with US exposure, both in vitro and in vivo. However, without US, only very low transfection level could be obtained in vivo. The DNA/PEI complex showed protective effect against digestion of DNase I enzymes as compared with groups without PEI or to which PEI was added following the mixing of plasmid DNA with DNase I. PEI enhanced the US transfection efficiency by increasing both the intracellular uptake of plasmid DNA and the percentage of transfected cells. Most of the DNA uptake occurred at 3h after US exposure, suggesting that endocytosis took place. Moreover, the PEI-facilitated US gene transfection depended on the ratio of PEI and DNA (N/P ratio), which was different for in-vitro and in-vivo conditions. This system could be applied in future human gene therapies. Copyright © 2012 Elsevier B.V. All rights reserved.

Role of cholesterol on the transfection barriers of cationic lipid/DNA complexes

NASA Astrophysics Data System (ADS)

Pozzi, Daniela; Cardarelli, Francesco; Salomone, Fabrizio; Marchini, Cristina; Amenitsch, Heinz; Barbera, Giorgia La; Caracciolo, Giulio

2014-08-01

Most lipid formulations need cholesterol for efficient transfection, but the precise motivation remains unclear. Here, we have investigated the effect of cholesterol on the transfection efficiency (TE) of cationic liposomes made of 1,2-dioleoyl-3-trimethylammonium-propane and dioleoylphosphocholine in Chinese hamster ovary cells. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by TE, synchrotron small angle X-ray scattering, and laser scanning confocal microscopy experiments. We prove that cholesterol-containing lipoplexes enter the cells using different endocytosis pathways. Formulations with high cholesterol content efficiently escape from endosomes and exhibit a lamellar-nonlamellar phase transition in mixture with biomembrane mimicking lipid formulations. This might explain both the DNA release ability and the high transfection efficiency. These studies highlight the enrichment in cholesterol as a decisive factor for transfection and will contribute to the rational design of lipid nanocarriers with superior TE.

Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation.

PubMed

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Witts, Emily C; Miles, Gareth B; Dholakia, Kishan; Gunn-Moore, Frank J

2013-11-21

A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided "point-and-transfect" user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits.

Enhanced gene transfection performance and biocompatibility of polyethylenimine through pseudopolyrotaxane formation with α-cyclodextrin.

PubMed

Hu, Li-Zhong; Wan, Ning; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

2017-03-24

Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.

Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay.

PubMed

Feltus, A; Grosvenor, A L; Conover, R C; Anderson, K W; Daunert, S

2001-04-01

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines

PubMed Central

Regla-Nava, Jose A.; Jimenez-Guardeño, Jose M.; Nieto-Torres, Jose L.; Gallagher, Thomas M.; Enjuanes, Luis; DeDiego, Marta L.

2013-01-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. PMID:23911968

Revealing the ability of a novel polysaccharide bioflocculant in bioremediation of heavy metals sensed in a Vibrio bioluminescence reporter assay.

PubMed

Sajayan, Arya; Seghal Kiran, G; Priyadharshini, S; Poulose, Navya; Selvin, Joseph

2017-09-01

A bioflocculant-producing bacterial strain, designated MSI021, was isolated from the marine sponge Dendrilla nigra and demonstrated 94% flocculation activity in a kaolin clay suspension. MSI021 was identified as Bacillus cereus based on phylogenetic affiliation and biochemical characteristics. The purified extra-cellular bioflocculant was chemically elucidated as a polysaccharide molecule. The polysaccharide bioflocculant was stable under both acidic and alkaline conditions (pH 2.0-10.0) and temperatures up to 100 °C. The purified bioflocculant efficiently nucleated the formation of silver nanoparticles which showed broad spectrum antibacterial activity. The ability of the bioflocculant to remediate heavy metal toxicity was evaluated by measuring the inhibition of bioluminescence expression in Vibrio harveyi. Enrichment of heavy metals such as zinc, mercury and copper at concentrations of 1, 2 and 3 mM in culture media showed significant reduction of bioluminescence in Vibrio, whereas media enriched with heavy metals and bioflocculant showed dose dependent improvement in the expression of bioluminescence. The assay results demonstrated that the polysaccharide bioflocculant effectively mitigates heavy metal toxicity, thereby improving the expression of bioluminescence in Vibrio. This bioluminescence reporter assay can be developed into a high-throughput format to monitor and evaluate of heavy metal toxicity. The findings of this study revealed that a novel polysaccharide bioflocculant produced by a marine B. cereus demonstrated strong flocculating performance and was effective in nucleating the formation antibacterial silver nanoparticles and removing heavy metals. These results suggest that the MSI021 polysaccharide bioflocculant can be used to develop greener waste water treatment systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells

PubMed Central

Pant, Kishor; Yadav, Ajay K.; Gupta, Parul; Rathore, Abhishek Singh; Nayak, Baibaswata; Venugopal, Senthil K.

2016-01-01

Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 μM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and β-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 μM and 5-fold with 100 μM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells. PMID:27708347

Proteome alteration induced by hTERT transfection of human fibroblast cells.

PubMed

Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

2008-04-17

Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair

Improving ultrasound gene transfection efficiency by controlling ultrasound excitation of microbubbles

PubMed Central

Fan, Z.; Chen, D.; Deng, C.X.

2013-01-01

Ultrasound application in the presence of microbubbles has shown great potential for non-viral gene transfection via transient disruption of cell membrane (sonoporation). However, improvement of its efficiency has largely relied on empirical approaches without consistent and translatable results. The goal of this study is to develop a rational strategy based on new results obtained using novel experimental techniques and analysis to improve sonoporation gene transfection. We conducted experiments using targeted microbubbles that were attached to cell membrane to facilitate sonoporation. We quantified the dynamic activities of microbubbles exposed to pulsed ultrasound and the resulting sonoporation outcome and identified distinct regimes of characteristic microbubble behaviors: stable cavitation, coalescence and translation, and inertial cavitation. We found that inertial cavitation generated the highest rate of membrane poration. By establishing direct correlation of ultrasound-induced bubble activities with intracellular uptake and pore size, we designed a ramped pulse exposure scheme for optimizing microbubble excitation to improve sonoporation gene transfection. We implemented a novel sonoporation gene transfection system using an aqueous two phase system (ATPS) for efficient use of reagents and high throughput operation. Using plasmid coding for the green fluorescence protein (GFP), we achieved a sonoporation transfection efficiency in rate aortic smooth muscle cells (RASMCs) of 6.9% ± 2.2% (n = 9), comparable with lipofection (7.5% ± 0.8%, n = 9). Our results reveal characteristic microbubble behaviors responsible for sonoporation and demonstrated a rational strategy to improve sonoporation gene transfection. PMID:23770009

Beyond D-luciferin: Expanding the Scope of Bioluminescence Imaging in vivo

PubMed Central

Adams, Spencer T.; Miller, Stephen C.

2014-01-01

The light-emitting chemical reaction catalyzed by the enzyme firefly luciferase is widely used for noninvasive imaging in live mice. However, photon emission from the luciferase is critically dependent on the chemical properties of its substrate, D-luciferin. In this review, we describe recent work to replace the natural luciferase substrate with synthetic analogs that extend the scope of bioluminescence imaging. PMID:25078002

Establishment of a stable transfection system for genetic manipulation of Babesia gibsoni.

PubMed

Liu, Mingming; Adjou Moumouni, Paul Franck; Asada, Masahito; Hakimi, Hassan; Masatani, Tatsunori; Vudriko, Patrick; Lee, Seung-Hun; Kawazu, Shin-Ichiro; Yamagishi, Junya; Xuan, Xuenan

2018-04-23

Genetic manipulation techniques, such as transfection, have been previously reported in many protozoan parasites. In Babesia, stable transfection systems have only been established for bovine Babesia parasites. We recently reported a transient transfection system and the selection of promoter candidates for Babesia gibsoni. The establishment of a stable transfection system for B. gibsoni is considered to be urgent to improve our understanding of the basic biology of canine Babesia parasites for a better control of babesiosis. GFP-expressing parasites were observed by fluorescence microscopy as early as two weeks after drug selection, and consistently expressed GFP for more than 3 months without drug pressure. Genome integration was confirmed by PCR, sequencing and Southern blot analysis. We present the first successful establishment of a stable transfection system for B. gibsoni. This finding will facilitate functional analysis of Babesia genomes using genetic manipulation and will serve as a foundation for the development of tick-Babesia and host-Babesia infection models.

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

PubMed

Endlich, B; Salavati, R; Sullivan, T; Ling, C C

1992-12-01

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression

Gene silencing of beta-catenin in melanoma cells retards their growth but promotes the formation of pulmonary metastasis in mice.

PubMed

Takahashi, Yuki; Nishikawa, Makiya; Suehara, Tetsuya; Takiguchi, Naomi; Takakura, Yoshinobu

2008-11-15

Altered expression of beta-catenin, a key component of the Wnt signaling pathway, is involved in a variety of cancers because increased levels of beta-catenin protein are frequently associated with enhanced cellular proliferation. Although our previous study demonstrated that gene silencing of beta-catenin in melanoma B16-BL6 cells by plasmid DNA (pDNA) expressing short-hairpin RNA targeting the gene (pshbeta-catenin) markedly suppressed their growth in vivo, gene silencing of beta-catenin could promote tumor metastasis by the rearranging cell adhesion complex. In this study, we investigated how silencing of beta-catenin affects metastatic aspects of melanoma cells. Transfection of B16-BL6 cells with pshbeta-catenin significantly reduced the amount of cadherin protein, a cell adhesion molecule binding to beta-catenin, with little change in its mRNA level. Cadherin-derived fragments were detected in culture media of B16-BL6 cells transfected with pshbeta-catenin, suggesting that cadherin is shed from the cell surface when the expression of beta-catenin is reduced. The mobility of B16-BL6 cells transfected with pshbeta-catenin was greater than that of cells transfected with any of the control pDNAs. B16-BL6 cells stably transfected with pshbeta-catenin (B16/pshbeta-catenin) formed less or an equal number of tumor nodules in the lung than cells stably transfected with other plasmids when injected into mice via the tail vein. However, when subcutaneously inoculated, B16/pshbeta-catenin cells formed more nodules in the lung than the other stably transfected cells. These results raise concerns about the gene silencing of beta-catenin for inhibiting tumor growth, because it promotes tumor metastasis by reducing the amount of cadherin in tumor cells. (c) 2008 Wiley-Liss, Inc.

A puzzling homology: a brittle star using a putative cnidarian-type luciferase for bioluminescence.

PubMed

Delroisse, Jérôme; Ullrich-Lüter, Esther; Blaue, Stefanie; Ortega-Martinez, Olga; Eeckhaut, Igor; Flammang, Patrick; Mallefet, Jérôme

2017-04-01

Bioluminescence relies on the oxidation of a luciferin substrate catalysed by a luciferase enzyme. Luciferins and luciferases are generic terms used to describe a large variety of substrates and enzymes. Whereas luciferins can be shared by phylogenetically distant organisms which feed on organisms producing them, luciferases have been thought to be lineage-specific enzymes. Numerous light emission systems would then have co-emerged independently along the tree of life resulting in a plethora of non-homologous luciferases. Here, we identify for the first time a candidate luciferase of a luminous echinoderm, the ophiuroid Amphiura filiformis Phylogenomic analyses identified the brittle star predicted luciferase as homologous to the luciferase of the sea pansy Renilla (Cnidaria), contradicting with the traditional viewpoint according to which luciferases would generally be of convergent origins. The similarity between the Renilla and Amphiura luciferases allowed us to detect the latter using anti- Renilla luciferase antibodies. Luciferase expression was specifically localized in the spines which were demonstrated to be the bioluminescent organs in vivo However, enzymes homologous to the Renilla luciferase but unable to trigger light emission were also identified in non-luminous echinoderms and metazoans. Our findings strongly indicate that those enzymes, belonging to the haloalkane dehalogenase family, might then have been convergently co-opted into luciferases in cnidarians and echinoderms. In these two benthic suspension-feeding species, similar ecological pressures would constitute strong selective forces for the functional shift of these enzymes and the emergence of bioluminescence. © 2017 The Authors.

A puzzling homology: a brittle star using a putative cnidarian-type luciferase for bioluminescence

PubMed Central

Ullrich-Lüter, Esther; Blaue, Stefanie; Ortega-Martinez, Olga; Eeckhaut, Igor; Flammang, Patrick; Mallefet, Jérôme

2017-01-01

Bioluminescence relies on the oxidation of a luciferin substrate catalysed by a luciferase enzyme. Luciferins and luciferases are generic terms used to describe a large variety of substrates and enzymes. Whereas luciferins can be shared by phylogenetically distant organisms which feed on organisms producing them, luciferases have been thought to be lineage-specific enzymes. Numerous light emission systems would then have co-emerged independently along the tree of life resulting in a plethora of non-homologous luciferases. Here, we identify for the first time a candidate luciferase of a luminous echinoderm, the ophiuroid Amphiura filiformis. Phylogenomic analyses identified the brittle star predicted luciferase as homologous to the luciferase of the sea pansy Renilla (Cnidaria), contradicting with the traditional viewpoint according to which luciferases would generally be of convergent origins. The similarity between the Renilla and Amphiura luciferases allowed us to detect the latter using anti-Renilla luciferase antibodies. Luciferase expression was specifically localized in the spines which were demonstrated to be the bioluminescent organs in vivo. However, enzymes homologous to the Renilla luciferase but unable to trigger light emission were also identified in non-luminous echinoderms and metazoans. Our findings strongly indicate that those enzymes, belonging to the haloalkane dehalogenase family, might then have been convergently co-opted into luciferases in cnidarians and echinoderms. In these two benthic suspension-feeding species, similar ecological pressures would constitute strong selective forces for the functional shift of these enzymes and the emergence of bioluminescence. PMID:28381628

What Orthopaedic Operating Room Surfaces Are Contaminated With Bioburden? A Study Using the ATP Bioluminescence Assay.

PubMed

Richard, Raveesh Daniel; Bowen, Thomas R

2017-07-01

Contaminated operating room surfaces can increase the risk of orthopaedic infections, particularly after procedures in which hardware implantation and instrumentation are used. The question arises as to how surgeons can measure surface cleanliness to detect increased levels of bioburden. This study aims to highlight the utility of adenosine triphosphate (ATP) bioluminescence technology as a novel technique in detecting the degree of contamination within the sterile operating room environment. What orthopaedic operating room surfaces are contaminated with bioburden? When energy is required for cellular work, ATP breaks down into adenosine biphosphate (ADP) and phosphate (P) and in that process releases energy. This process is inherent to all living things and can be detected as light emission with the use of bioluminescence assays. On a given day, six different orthopaedic surgery operating rooms (two adult reconstruction, two trauma, two spine) were tested before surgery with an ATP bioluminescence assay kit. All of the cases were considered clean surgery without infection, and this included the previously performed cases in each sampled room. These rooms had been cleaned and prepped for surgery but the patients had not been physically brought into the room. A total of 13 different surfaces were sampled once in each room: the operating room (OR) preparation table (both pre- and postdraping), OR light handles, Bovie machine buttons, supply closet countertops, the inside of the Bair Hugger™ hose, Bair Hugger™ buttons, right side of the OR table headboard, tourniquet machine buttons, the Clark-socket attachment, and patient positioners used for total hip and spine positioning. The relative light units (RLUs) obtained from each sample were recorded and data were compiled and averaged for analysis. These values were compared with previously published ATP benchmark values of 250 to 500 RLUs to define cleanliness in both the hospital and restaurant industries. All

Analysis of Fleet Reports of Bioluminescence in the Indian Ocean

DTIC Science & Technology

1981-12-14

Kalle [ 20,21], who theorized that they were caused by seismic disturbances. According to his theory, such disturbances would produce wheels in...Whether this phenomenon is the same as that described by Kalle is uncertain. The description of the phosphorescent wheel, however, was truly classic. The...Luciferin in Some Shallow-Water FisheF Comp. Biochem. Physiol. 40A:163-179, 1971 . 14. P. J. Herring, "Observations of Bioluminescence at Sea," Mar. Obs. 46

HTLV-1 Tax Mediated Downregulation of miRNAs Associated with Chromatin Remodeling Factors in T Cells with Stably Integrated Viral Promoter

PubMed Central

Rahman, Saifur; Quann, Kevin; Pandya, Devanshi; Singh, Shruti; Khan, Zafar K.; Jain, Pooja

2012-01-01

RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type. PMID:22496815

UV and arsenate toxicity: a specific and sensitive yeast bioluminescence assay.

PubMed

Bakhrat, Anya; Eltzov, Evgeni; Finkelstein, Yishay; Marks, Robert S; Raveh, Dina

2011-06-01

We describe a Saccharomyces cerevisiae bioluminescence assay for UV and arsenate in which bacterial luciferase genes are regulated by the promoter of the yeast gene, UFO1. UFO1 encodes the F-box subunit of the Skp1–Cdc53–F-box protein ubiquitin ligase complex and is induced by DNA damage and by arsenate. We engineered the UFO1 promoter into an existing yeast bioreporter that employs human genes for detection of steroid hormone-disrupting compounds in water bodies. Our analysis indicates that use of an endogenous yeast promoter in different mutant backgrounds allows discrimination between different environmental signals. The UFO1-engineered yeast give a robust bioluminescence response to UVB and can be used for evaluating UV protective sunscreens. They are also effective in detecting extremely low concentrations of arsenate, particularly in pdr5Δ mutants that lack a mechanism to extrude toxic chemicals; however, they do not respond to cadmium or mercury. Combined use of endogenous yeast promoter elements and mutants of stress response pathways may facilitate development of high-specificity yeast bioreporters able to discriminate between closely related chemicals present together in the environment.

Bioluminescence imaging of c-fos gene expression accompanying filial imprinting in the newly hatched chick brain.

PubMed

Yamaguchi, Shinji; Iikubo, Eiji; Hirose, Naoki; Kitajima, Takaaki; Katagiri, Sachiko; Kawamori, Ai; Fujii-Taira, Ikuko; Matsushima, Toshiya; Homma, Koichi J

2010-06-01

Bioluminescence imaging is a powerful tool for examining gene expression in living animals. Previously, we reported that exogenous DNA could be successfully delivered into neurons in the newly hatched chick brain using electroporation. Here, we show the in vivo bioluminescence imaging of c-fos promoter activity and its upregulation, which is associated with filial imprinting. The upregulation of c-fos gene expression correlated with both the strength of the chicks' approach activity to the training object and the acquisition of memory. The present technique should be a powerful tool for analyzing the time changes in neural activity of certain brain areas in real-time during memory formation, using brains of living animals.

A new bioluminescent reporter system to study the biodistribution of systematically injected tumor-derived bioluminescent extracellular vesicles in mice

PubMed Central

Gangadaran, Prakash; Li, Xiu Juan; Lee, Ho Won; Oh, Ji Min; Kalimuthu, Senthilkumar; Rajendran, Ramya Lakshmi; Son, Seung Hyun; Baek, Se Hwan; Singh, Thoudam Debraj; Zhu, Liya; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

2017-01-01

In vivo biodistribution and fate of extracellular vesicles (EVs) are still largely unknown and require reliable in vivo tracking techniques. In this study, in vivo bioluminescence imaging (BLI) using Renilla luciferase (Rluc) was developed and applied to monitoring of EVs derived from thyroid cancer (CAL-62 cells) and breast cancer (MDA-MB-231) in nude mice after intravenous administration and was compared with a dye-based labeling method for EV derived from CAL-62 cells. The EVs were successfully labeled with Rluc and visualized by BLI in mice. In vivo distribution of the EVs, as measured by BLI, was consistent with the results of ex vivo organ analysis. EV-CAL-62/Rluc showed strong signals at lung followed by liver, spleen & kidney (P < 0.05). EV-MDA-MB-231/Rluc showed strong signals at liver followed by lung, spleen & kidney (P < 0.05). EV-CAL-62/Rluc and EV-MDA-MB-231/Rluc stayed in animal till day 9 and 3, respectively; showed a differential distribution. Spontaneous EV-CAL-62/Rluc shown distributed mostly to lung followed by liver, spleen & kidney. The new BLI system used to show spontaneous distribution of EV-CAL-62/Rluc in subcutaneous CAL-62/Rluc bearing mice. Dye (DiR)-labeled EV-CAL-62/Rluc showed a different distribution in vivo & ex vivo compared to EV-CAL-62/Rluc. Fluorescent signals were predominately detected in the liver (P < 0.05) and spleen (P < 0.05) regions. The bioluminescent EVs developed in this study may be used for monitoring of EVs in vivo. This novel reporter-imaging approach to visualization of EVs in real time is expected to pave the way for monitoring of EVs in EV-based treatments. PMID:29299117

Nucleic acid detection using BRET-beacons based on bioluminescent protein-DNA hybrids.

PubMed

Engelen, Wouter; van de Wiel, Kayleigh M; Meijer, Lenny H H; Saha, Bedabrata; Merkx, Maarten

2017-03-02

Bioluminescent molecular beacons have been developed using a modular design approach that relies on BRET between the bright luciferase NanoLuc and a Cy3 acceptor. While classical molecular beacons are hampered by background fluorescence and scattering, these BRET-beacons allow detection of low pM concentrations of nucleic acids directly in complex media.

Adenosine triphosphate bioluminescence for bacteriological surveillance and reprocessing strategies for minimizing risk of infection transmission by duodenoscopes

PubMed Central

Sethi, Saurabh; Huang, Robert J.; Barakat, Monique T.; Banaei, Niaz; Friedland, Shai; Banerjee, Subhas

2017-01-01

Background/Aims Recent outbreaks of duodenoscope-transmitted infections underscore the importance of adequate endoscope reprocessing. Adenosine triphosphate (ATP) bioluminescence testing allows rapid evaluation of endoscopes for bacteriological/biological residue. In this prospective study we evaluate the utility of ATP in bacteriological surveillance, and the effects of endoscopy staff education and dual cycles of cleaning and high-level disinfection (HLD) on endoscope reprocessing. Methods ATP bioluminescence was measured after pre-cleaning, manual cleaning and HLD on rinsates from suction-biopsy channels of all endoscopes and elevator channels of duodenoscopes/linear echoendoscopes after use. ATP bioluminescence was re-measured in duodenoscopes (1) after re-education and competency testing of endoscopy staff, and subsequently (2) after 2 cycles of pre-cleaning and manual cleaning and single cycle of HLD, or (3) after 2 cycles of pre-cleaning, manual cleaning and HLD. Results The ideal ATP bioluminescence benchmark of <200 relative light units (RLUs) after manual cleaning was achieved from suction-biopsy channel rinsates of all endoscopes, but 9 of 10 duodenoscope elevator channel rinsates failed to meet this benchmark. Re-education reduced RLUs in duodenoscope elevator channel rinsates after pre-cleaning (23218.0 vs 1340.5 RLUs, p<0.01) and HLD (177.0 vs 12.0 RLUs, p<0.01). After 2 cycles of manual cleaning/HLD, duodenoscope elevator channel RLUs achieved levels similar to sterile water, with corresponding negative cultures. Conclusions ATP testing offers a rapid, inexpensive alternative for detection of endoscope microbial residue. Re-education of endoscopy staff and 2 cycles of cleaning and HLD decrease elevator channel RLUs to levels similar to sterile water and may therefore minimize the risk of transmission of infections by duodenoscopes. PMID:27818222

Comparisons of hybrid radiosity-diffusion model and diffusion equation for bioluminescence tomography in cavity cancer detection

NASA Astrophysics Data System (ADS)

Chen, Xueli; Yang, Defu; Qu, Xiaochao; Hu, Hao; Liang, Jimin; Gao, Xinbo; Tian, Jie

2012-06-01

Bioluminescence tomography (BLT) has been successfully applied to the detection and therapeutic evaluation of solid cancers. However, the existing BLT reconstruction algorithms are not accurate enough for cavity cancer detection because of neglecting the void problem. Motivated by the ability of the hybrid radiosity-diffusion model (HRDM) in describing the light propagation in cavity organs, an HRDM-based BLT reconstruction algorithm was provided for the specific problem of cavity cancer detection. HRDM has been applied to optical tomography but is limited to simple and regular geometries because of the complexity in coupling the boundary between the scattering and void region. In the provided algorithm, HRDM was first applied to three-dimensional complicated and irregular geometries and then employed as the forward light transport model to describe the bioluminescent light propagation in tissues. Combining HRDM with the sparse reconstruction strategy, the cavity cancer cells labeled with bioluminescent probes can be more accurately reconstructed. Compared with the diffusion equation based reconstruction algorithm, the essentiality and superiority of the HRDM-based algorithm were demonstrated with simulation, phantom and animal studies. An in vivo gastric cancer-bearing nude mouse experiment was conducted, whose results revealed the ability and feasibility of the HRDM-based algorithm in the biomedical application of gastric cancer detection.

Comparisons of hybrid radiosity-diffusion model and diffusion equation for bioluminescence tomography in cavity cancer detection.

PubMed

Chen, Xueli; Yang, Defu; Qu, Xiaochao; Hu, Hao; Liang, Jimin; Gao, Xinbo; Tian, Jie

2012-06-01

Bioluminescence tomography (BLT) has been successfully applied to the detection and therapeutic evaluation of solid cancers. However, the existing BLT reconstruction algorithms are not accurate enough for cavity cancer detection because of neglecting the void problem. Motivated by the ability of the hybrid radiosity-diffusion model (HRDM) in describing the light propagation in cavity organs, an HRDM-based BLT reconstruction algorithm was provided for the specific problem of cavity cancer detection. HRDM has been applied to optical tomography but is limited to simple and regular geometries because of the complexity in coupling the boundary between the scattering and void region. In the provided algorithm, HRDM was first applied to three-dimensional complicated and irregular geometries and then employed as the forward light transport model to describe the bioluminescent light propagation in tissues. Combining HRDM with the sparse reconstruction strategy, the cavity cancer cells labeled with bioluminescent probes can be more accurately reconstructed. Compared with the diffusion equation based reconstruction algorithm, the essentiality and superiority of the HRDM-based algorithm were demonstrated with simulation, phantom and animal studies. An in vivo gastric cancer-bearing nude mouse experiment was conducted, whose results revealed the ability and feasibility of the HRDM-based algorithm in the biomedical application of gastric cancer detection.

EXPERIMENTS ON STABLY AND NEUTRALLY STRATIFIED FLOW OVER A MODEL THREE-DIMENSIONAL HILL

EPA Science Inventory

The flow structure over a bell shaped hill (reciprocal of a fourth order polynomial in cross section and height h) was studied in large and small stably stratified towing tanks (with uniform density gradients) and in an unstratified wind tunnel. Observations were made at Froude n...

Guide RNA selection for CRISPR-Cas9 transfections in Plasmodium falciparum.

PubMed

Ribeiro, Jose M; Garriga, Meera; Potchen, Nicole; Crater, Anna K; Gupta, Ankit; Ito, Daisuke; Desai, Sanjay A

2018-06-12

CRISPR-Cas9 mediated genome editing is addressing key limitations in the transfection of malaria parasites. While this method has already simplified the needed molecular cloning and reduced the time required to generate mutants in the human pathogen Plasmodium falciparum, optimal selection of required guide RNAs and guidelines for successful transfections have not been well characterized, leading workers to use time-consuming trial and error approaches. We used a genome-wide computational approach to create a comprehensive and publicly accessible database of possible guide RNA sequences in the P. falciparum genome. For each guide, we report on-target efficiency and specificity scores as well as information about the genomic site relevant to optimal design of CRISPR-Cas9 transfections to modify, disrupt, or conditionally knockdown any gene. As many antimalarial drug and vaccine targets are encoded by multigene families, we also developed a new paralog specificity score that should facilitate modification of either a single family member of interest or multiple paralogs that serve overlapping roles. Finally, we tabulated features of successful transfections in our laboratory, providing broadly useful guidelines for parasite transfections. Molecular studies aimed at understanding parasite biology or characterizing drug and vaccine targets in P. falciparum should be facilitated by this comprehensive database. Published by Elsevier Ltd.

Bioanalytical Applications of Real-Time ATP Imaging Via Bioluminescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruenhagen, Jason Alan

The research discussed within involves the development of novel applications of real-time imaging of adenosine 5'-triphosphate (ATP). ATP was detected via bioluminescence and the firefly luciferase-catalyzed reaction of ATP and luciferin. The use of a microscope and an imaging detector allowed for spatially resolved quantitation of ATP release. Employing this method, applications in both biological and chemical systems were developed. First, the mechanism by which the compound 48/80 induces release of ATP from human umbilical vein endothelial cells (HUVECs) was investigated. Numerous enzyme activators and inhibitors were utilized to probe the second messenger systems involved in release. Compound 48/80 activatedmore » a G{sub q}-type protein to initiate ATP release from HUVECs. Ca 2+ imaging along with ATP imaging revealed that activation of phospholipase C and induction of intracellular Ca 2+ signaling were necessary for release of ATP. Furthermore, activation of protein kinase C inhibited the activity of phospholipase C and thus decreased the magnitude of ATP release. This novel release mechanism was compared to the existing theories of extracellular release of ATP. Bioluminescence imaging was also employed to examine the role of ATP in the field of neuroscience. The central nervous system (CNS) was dissected from the freshwater snail Lymnaea stagnalis. Electrophysiological experiments demonstrated that the neurons of the Lymnaea were not damaged by any of the components of the imaging solution. ATP was continuously released by the ganglia of the CNS for over eight hours and varied from ganglion to ganglion and within individual ganglia. Addition of the neurotransmitters K + and serotonin increased release of ATP in certain regions of the Lymnaea CNS. Finally, the ATP imaging technique was investigated for the study of drug release systems. MCM-41-type mesoporous nanospheres were loaded with ATP and end-capped with mercaptoethanol functionalized Cd

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs.

PubMed

Khan, Aly A; Betel, Doron; Miller, Martin L; Sander, Chris; Leslie, Christina S; Marks, Debora S

2009-06-01

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

Plaque retention by self-ligating vs elastomeric orthodontic brackets: quantitative comparison of oral bacteria and detection with adenosine triphosphate-driven bioluminescence.

PubMed

Pellegrini, Peter; Sauerwein, Rebecca; Finlayson, Tyler; McLeod, Jennifer; Covell, David A; Maier, Tom; Machida, Curtis A

2009-04-01

Enamel decalcification is a common problem in orthodontics. The objectives of this randomized clinical study were to enumerate and compare plaque bacteria surrounding 2 bracket types, self-ligating (SL) vs elastomeric ligating (E), and to determine whether adenosine triphosphate (ATP)-driven bioluminescence could be used for rapid assessment of bacterial load in plaque. Patients (ages, 11-17 years) were bonded with SL and E brackets in 14 maxillary and 12 mandibular arches by using a split-mouth design. Recall visits were at 1 and 5 weeks after bonding. Plaque specimens were assayed for oral bacteria and subjected to ATP-driven bioluminescence determinations with a luciferin-based assay. In most patients, teeth bonded with SL attachments had fewer bacteria in plaque than did teeth bonded with E brackets. At 1 and 5 weeks after bonding, the means for SL vs E brackets were statistically lower for total bacteria and oral streptococci (P <0.05). ATP bioluminescence values were statistically correlated to the total oral bacteria and oral streptococci, with correlation coefficients of 0.895 and 0.843, respectively. SL appliances promote reduced retention of oral bacteria, and ATP bioluminescence might be a useful tool in the rapid quantification of bacterial load and the assessment of oral hygiene during orthodontic treatment.

Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line

PubMed Central

Salimzadeh, Loghman; Jaberipour, Mansooreh; Hosseini, Ahmad; Ghaderi, Abbas

2013-01-01

Background Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. PMID:23799175

Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production.

PubMed

Cervera, Laura; Kamen, Amine A

2018-01-01

Large-scale transient transfection of mammalian cell suspension cultures enables the production of biological products in sufficient quantity and under stringent quality attributes to perform accelerated in vitro evaluations and has the potential to support preclinical or even clinical studies. Here we describe the methodology to produce VLPs in a 3L bioreactor, using suspension HEK 293 cells and PEIPro as a transfection reagent. Cells are grown in the bioreactor to 1 × 10 6 cells/mL and transfected with a plasmid DNA-PEI complex at a ratio of 1:2. Dissolved oxygen and pH are controlled and are online monitored during the production phase and cell growth and viability can be measured off line taking samples from the bioreactor. If the product is labeled with a fluorescent marker, transfection efficiency can be also assessed using flow cytometry analysis. Typically, the production phase lasts between 48 and 96 h until the product is harvested.

The Synergistic Effect between Electrical and Chemical Factors in Plasma Gene/Molecule-Transfection

NASA Astrophysics Data System (ADS)

Jinno, Masafumi

2016-09-01

This study has been done to know what kind of factors in plasma and processes on cells promote plasma gene/molecule transfection. We have discovered a new plasma source using a microcapillary electrode which enables high transfection efficiency and high cell survivability simultaneously. However, the mechanism of the transfection by plasma was not clear. To clarify the transfection mechanisms by micro plasma, we focused on the effects of electrical (current, charge, field, etc.) and chemical (radicals, RONS, etc.) factors generated by the micro plasma and evaluated the contribution weight of three groups of the effects and processes, i.e. electrical, chemical and biochemical ones. At first, the necessity of the electrical factors was estimated by the laser produced plasma (LPP). Mouse L-929 fibroblast cell was cultured on a 96-well plate or 12-well micro slide chamber. Plasmids pCX-EGFP in Tris-EDTA buffer was dropped on the cells and they were exposed to the capillary discharge plasma (CDP) or the LPP. In the case of the CDP, the plasma was generated between the tip of the capillary electrode and the cells so that both electrical and chemical factors were supplied to the cells. In this setup, about 20% of average transfection efficiency was obtained. In the case of the LPP, the plasma was generated apart from the cells so that electrical factors were not supplied to the cells. In this setup, no transfection was observed. These results show that the electrical factors are necessary for the plasma gene transfection. Next, the necessity of the chemical factors was estimated the effect of catalase to remove H2O2 in CDP. The transfection efficiency decreased to 0.4 by scavenging H2O2 with catalase. However, only the solution of H2O2 caused no gene transfection in cells. These results shows that H2O2 is important species to cause gene/molecule transfection but still needs a synergistic effect with electrical or other chemical factors. This work was partly supported by

Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation

NASA Astrophysics Data System (ADS)

Creasey, Rhiannon; Hook, Andrew; Thissen, Helmut; Voelcker, Nicolas H.

2007-12-01

Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing [1, 2]. Low transfection efficiency has so far limited the application and utility of this technique. Recently, the transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA array resulting in the electroporation of cells attached to the surface [3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption of attached DNA, increasing the concentration of DNA available for cellular uptake [5]. In the present study, the optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the microarray

Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation

PubMed Central

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Witts, Emily C.; Miles, Gareth B.; Dholakia, Kishan; Gunn-Moore, Frank J.

2013-01-01

A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided “point-and-transfect” user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits. PMID:24257461

[The effect of retrovirus-mediated hTRT transfection into cultured oral keratinocytes].

PubMed

Huang, Ji-yan; Liu, Wei; Zhou, Zeng-tong; Zhou, Hai-wen

2014-06-01

Human telomerase reverse transcriptase (hTRT) was transfected into cultured oral keratinocytes (OKC) mediated by pBABE-tert recombined retrovirus to investigate the effect on OKC lifespan. pBABE-tert recombined retrovirus loaded with hTRT gene was amplified by transfected PT67 cells, and then transfected into cultured OKC in vitro. The positive clones of OKC were separated by puromycin and subcultured. Telomerase activity was analyzed by telomerase PCR-ELISA and PCR-PAGE. The hTRT positive clones of OKC showed telomerase expression, with extending lifespan to 8-9 passages. The hTRT transfected OKC can prolong doubly lifespan but not be immortalized, which indicates that cellular immortality mechanism is complicated and multi-controled. Telomerase activity is the key for cell immortalization but not the only impact factor.

Whole-cell bioluminescent bioreporter sensing of foodborne toxicants

NASA Astrophysics Data System (ADS)

Ripp, Steve A.; Applegate, Bruce M.; Simpson, Michael L.; Sayler, Gary S.

2001-03-01

The presence of biologically derived toxins in foods is of utmost significance to food safety and human health concerns. Biologically active amines, referred to as biogenic amines, serve as a noteworthy example, having been implicated as the causative agent in numerous food poisoning episodes. Of the various biogenic amines encountered, histamine, putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, spermine, and spermidine are considered to be the most significant, and can be used as hygienic-quality indicators of food. Biogenic amines can be monitored using whole-cell bioluminescent bioreporters, which represent a family of genetically engineered microorganisms that generate visible light in response to specific chemical or physical agents in their environment. The light response occurs due to transcriptional activation of a genetically incorporated lux cassette, and can be measured using standard photomultiplier devices. We have successfully engineered a lux-based bioreporter capable of detecting and monitoring the biogenic amine beta-phenylethylamine. This research represents a biologically-based sensor technology that can be readily integrated into Hazard Analysis Critical Control Point programs to provide a rugged monitoring regime that can be uniformly applied for field-based and in-house laboratory quality control analyses. Since the bioreporter and biosensing elements are completely self-contained within the sensor design, this system provides ease of use, with operational capabilities realized by simply combining the food sample with the bioreporter and allowing the sensor to process the ensuing bioluminescent signal and communicate the results. The application of this technology to the critically important issue of food safety and hygienic quality represents a novel method for detecting, monitoring, and preventing biologically active toxins in food commodities.

Bioluminescent luciferase-modified magnetic nanoparticles as potential imaging agents for mammalian spermatozoa detection and tracking

USDA-ARS?s Scientific Manuscript database

Background: Nanoparticles have emerged as key materials for developing applications in nanomedicine, nanobiotechnology, bioimaging and theranostics. Existing bioimaging technologies include bioluminescent resonance energy transfer-conjugated quantum dots (BRET-QDs). Despite the current use of BRET-Q...

Data-based mathematical modeling of vectorial transport across double-transfected polarized cells.

PubMed

Bartholomé, Kilian; Rius, Maria; Letschert, Katrin; Keller, Daniela; Timmer, Jens; Keppler, Dietrich

2007-09-01

Vectorial transport of endogenous small molecules, toxins, and drugs across polarized epithelial cells contributes to their half-life in the organism and to detoxification. To study vectorial transport in a quantitative manner, an in vitro model was used that includes polarized MDCKII cells stably expressing the recombinant human uptake transporter OATP1B3 in their basolateral membrane and the recombinant ATP-driven efflux pump ABCC2 in their apical membrane. These double-transfected cells enabled mathematical modeling of the vectorial transport of the anionic prototype substance bromosulfophthalein (BSP) that has frequently been used to examine hepatobiliary transport. Time-dependent analyses of (3)H-labeled BSP in the basolateral, intracellular, and apical compartments of cells cultured on filter membranes and efflux experiments in cells preloaded with BSP were performed. A mathematical model was fitted to the experimental data. Data-based modeling was optimized by including endogenous transport processes in addition to the recombinant transport proteins. The predominant contributions to the overall vectorial transport of BSP were mediated by OATP1B3 (44%) and ABCC2 (28%). Model comparison predicted a previously unrecognized endogenous basolateral efflux process as a negative contribution to total vectorial transport, amounting to 19%, which is in line with the detection of the basolateral efflux pump Abcc4 in MDCKII cells. Rate-determining steps in the vectorial transport were identified by calculating control coefficients. Data-based mathematical modeling of vectorial transport of BSP as a model substance resulted in a quantitative description of this process and its components. The same systems biology approach may be applied to other cellular systems and to different substances.

Rapid bacteriological screening of cosmetic raw materials by using bioluminescence.

PubMed

Nielsen, P; Van Dellen, E

1989-01-01

Incoming cosmetic raw materials are routinely tested for microbial content. Standard plate count methods require up to 72 h. A rapid, sensitive, and inexpensive raw material screening method was developed that detects the presence of bacteria by means of ATP (bioluminescence). With a 24-h broth enrichment, the minimum bacterial ATP detection threshold of 1 cfu/g sample can be achieved using purified firefly luciferin-luciferase and an ATP releasing reagent. By using this rapid screen, microbiologically free material may be released for production within 24 h, while contaminated material undergoes further quantitative and identification testing. In order for a raw material to be validated for this method it must be evaluated for (1) a potential nonmicrobial light-contributing reaction resulting in a false positive or, (2) degradation of the ATP giving a false negative, and (3) confirmation that the raw material has not overwhelmed the buffering capacity of the enrichment broth. The key criteria for a rapid screen was the sensitivity to detect less than one colony forming unit per g product, the speed to do this within 24 h, and cost efficiency. Bioluminescence meets these criteria. With an enrichment step, it can detect less than one cfu/g sample. After the enrichment step, analysis time per sample is approximately 2 min and the cost for material and reagents is less than one dollar per sample.

A look at some systemic properties of self-bioluminescent emission

NASA Astrophysics Data System (ADS)

Creath, Katherine

2008-08-01

Self-bioluminescent emission (SBE) is a type of biological chemiluminescence where photons are emitted as part of chemical reactions occurring during metabolic processes. This emission is also known as biophoton emission, ultraweak photon emission and ultraweak bioluminescence. This paper outlines research over the past century on some systemic properties of SBE as measured with biological detectors, photomultiplier detectors and ultra-sensitive imaging arrays. There is an apparent consensus in the literature that emission in the deep blue and ultraviolet (150-450nm) is related to DNA / RNA processes while emission in the red and near infrared (600-1000nm) is related to mitochondria and oxidative metabolisms involving reactive oxygen species, singlet oxygen and free radicals in plant, animal and human cells along with chlorophyll fluorescent decay in plants. Additionally, there are trends showing that healthy, unstressed and uninjured samples have less emission than samples that are unhealthy, stressed or injured. Mechanisms producing this emission can be narrowed down by isolating the wavelength region of interest and waiting for short-term fluorescence to decay leaving the ultraweak long-term metabolic emission. Examples of imaging this emission in healthy versus unhealthy, stressed versus unstressed, and injured versus uninjured plant parts are shown. Further discussion poses questions still to be answered related to properties such as coherence, photon statistics, and methodological means of isolating mechanisms.

Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.

PubMed

Green, David W; Kim, Eun-Jung; Jung, Han-Sung

2015-09-01

The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems. © 2015 Wiley Periodicals, Inc.

Adenosine triphosphate bioluminescence for bacteriologic surveillance and reprocessing strategies for minimizing risk of infection transmission by duodenoscopes.

PubMed

Sethi, Saurabh; Huang, Robert J; Barakat, Monique T; Banaei, Niaz; Friedland, Shai; Banerjee, Subhas

2017-06-01

Recent outbreaks of duodenoscope-transmitted infections underscore the importance of adequate endoscope reprocessing. Adenosine triphosphate (ATP) bioluminescence testing allows rapid evaluation of endoscopes for bacteriologic/biologic residue. In this prospective study we evaluate the utility of ATP in bacteriologic surveillance and the effects of endoscopy staff education and dual cycles of cleaning and high-level disinfection (HLD) on endoscope reprocessing. ATP bioluminescence was measured after precleaning, manual cleaning, and HLD on rinsates from suction-biopsy channels of all endoscopes and elevator channels of duodenoscopes/linear echoendoscopes after use. ATP bioluminescence was remeasured in duodenoscopes (1) after re-education and competency testing of endoscopy staff and subsequently (2) after 2 cycles of precleaning and manual cleaning and single cycle of HLD or (3) after 2 cycles of precleaning, manual cleaning, and HLD. The ideal ATP bioluminescence benchmark of <200 relative light units (RLUs) after manual cleaning was achieved from suction-biopsy channel rinsates of all endoscopes, but 9 of 10 duodenoscope elevator channel rinsates failed to meet this benchmark. Re-education reduced RLUs in duodenoscope elevator channel rinsates after precleaning (23,218.0 vs 1340.5 RLUs, P < .01) and HLD (177.0 vs 12.0 RLUs, P < .01). After 2 cycles of manual cleaning/HLD, duodenoscope elevator channel RLUs achieved levels similar to sterile water, with corresponding negative cultures. ATP testing offers a rapid, inexpensive alternative for detection of endoscope microbial residue. Re-education of endoscopy staff and 2 cycles of cleaning and HLD decreased elevator channel RLUs to levels similar to sterile water and may therefore minimize the risk of transmission of infections by duodenoscopes. Copyright © 2017 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

Mitrocomin from the jellyfish Mitrocoma cellularia with deleted C-terminal tyrosine reveals a higher bioluminescence activity compared to wild type photoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burakova, Ludmila P.; Natashin, Pavel V.; Markova, Svetlana V.

The full-length cDNA genes encoding five new isoforms of Ca2 +-regulated photoprotein mitrocomin from a small tissue sample of the outer bell margin containing photocytes of only one specimen of the luminous jellyfish Mitrocoma cellularia were cloned, sequenced, and characterized after their expression in Escherichia coli and subsequent purification. The analysis of cDNA nucleotide sequences encoding mitrocomin isoforms allowed suggestion that two isoforms might be the products of two allelic genes differing in one amino acid residue (64R/Q) whereas other isotypes appear as a result of transcriptional mutations. In addition, the crystal structure of mitrocomin was determined at 1.30 Åmore » resolution which expectedly revealed a high similarity with the structures of other hydromedusan photoproteins. Although mitrocomin isoforms reveal a high degree of identity of amino acid sequences, they vary in specific bioluminescence activities. At that, all isotypes displayed the identical bioluminescence spectra (473–474 nm with no shoulder at 400 nm). Fluorescence spectra of Ca2 +-discharged mitrocomins were almost identical to their light emission spectra similar to the case of Ca2 +-discharged aequorin, but different from Ca2 +-discharged obelins and clytin which fluorescence is red-shifted by 25–30 nm from bioluminescence spectra. The main distinction of mitrocomin from other hydromedusan photoproteins is an additional Tyr at the C-terminus. Using site-directed mutagenesis, we showed that this Tyr is not important for bioluminescence because its deletion even increases specific activity and efficiency of apo-mitrocomin conversion into active photoprotein, in contrast to C-terminal Pro of other photoproteins. Since genes in a population generally exist as different isoforms, it makes us anticipate the cloning of even more isoforms of mitrocomin and other hydromedusan photoproteins with different bioluminescence properties.« less

Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level

PubMed Central

Baubet, Valérie; Le Mouellic, Hervé; Campbell, Anthony K.; Lucas-Meunier, Estelle; Fossier, Philippe; Brûlet, Philippe

2000-01-01

Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca2+ stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca2+ increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals. PMID:10860991

Self-illuminating in vivo lymphatic imaging using a bioluminescence resonance energy transfer quantum dot nano-particle.

PubMed

Kosaka, Nobuyuki; Mitsunaga, Makoto; Bhattacharyya, Sukanta; Miller, Steven C; Choyke, Peter L; Kobayashi, Hisataka

2011-01-01

Autofluorescence arising from normal tissues can compromise the sensitivity and specificity of in vivo fluorescence imaging by lowering the target-to-background signal ratio. Since bioluminescence resonance energy transfer quantum dot (BRET-QDot) nano-particles can self-illuminate in near-infrared in the presence of the substrate, coelenterazine, without irradiating excitation lights, imaging using BRET-QDots does not produce any autofluorescence. In this study, we applied this BRET-QDot nano-particle to the in vivo lymphatic imaging in mice in order to compare with BRET, fluorescence or bioluminescence lymphatic imaging. BRET-QDot655, in which QDot655 is contained as a core, was injected at different sites (e.g. chin, ear, forepaws and hind paws) in mice followed by the intravenous coelenterazine injection, and then bioluminescence and fluorescence imaging were serially performed. In all mice, each lymphatic basin was clearly visualized in the BRET imaging with minimal background signals. The BRET signal in the lymph nodes lasted at least 30 min after coelenterazine injections. Furthermore, the BRET signal demonstrated better quantification than the fluorescence signal emitting from QDot655, the core of this BRET particle. These advantages of BRET-QDot allowed us to perform real-time, quantitative lymphatic imaging without image processing. BRET-Qdots have the potential to be a robust nano-material platform for developing optical molecular imaging probes. Copyright © 2010 John Wiley & Sons, Ltd.

Self-illuminating in vivo lymphatic imaging using a bioluminescence resonance energy transfer quantum dot nano-particle

PubMed Central

Kosaka, Nobuyuki; Mitsunaga, Makoto; Bhattacharyya, Sukanta; Miller, Steven C.; Choyke, Peter L.; Kobayashi, Hisataka

2012-01-01

Autofluorescence arising from normal tissues can compromise the sensitivity and specificity of in vivo fluorescence imaging by lowering the target-to-background signal ratio. Since bioluminescence resonance energy transfer quantum dot (BRET-QDot) nano-particles can self-illuminate in near-infrared in the presence of the substrate, coelenterazine, without irradiating excitation lights, imaging using BRET-QDots does not produce any autofluorescence. In this study, we applied this BRET-QDot nano-particle to the in vivo lymphatic imaging in mice in order to compare with BRET, fluorescence or bioluminescence lymphatic imaging. BRET-QDot655, in which QDot655 is contained as a core, was injected at different sites (e.g. chin, ear, forepaws and hind paws) in mice followed by the intravenous coelenterazine injection, and then bioluminescence and fluorescence imaging were serially performed. In all mice, each lymphatic basin was clearly visualized in the BRET imaging with minimal background signals. The BRETsignal in the lymph nodes lasted at least 30 min after coelenterazine injections. Furthermore, the BRETsignal demonstrated better quantification than the fluorescence signal emitting from QDot655, the core of this BRET particle. These advantages of BRET-QDot allowed us to perform real-time, quantitative lymphatic imaging without image processing. BRET-Qdots have the potential to be a robust nano-material platform for developing optical molecular imaging probes. PMID:21351373

Assessment of Chitosan-Affected Metabolic Response by Peroxisome Proliferator-Activated Receptor Bioluminescent Imaging-Guided Transcriptomic Analysis

PubMed Central

Kao, Chia-Hung; Hsiang, Chien-Yun; Ho, Tin-Yun

2012-01-01

Chitosan has been widely used in food industry as a weight-loss aid and a cholesterol-lowering agent. Previous studies have shown that chitosan affects metabolic responses and contributes to anti-diabetic, hypocholesteremic, and blood glucose-lowering effects; however, the in vivo targeting sites and mechanisms of chitosan remain to be clarified. In this study, we constructed transgenic mice, which carried the luciferase genes driven by peroxisome proliferator-activated receptor (PPAR), a key regulator of fatty acid and glucose metabolism. Bioluminescent imaging of PPAR transgenic mice was applied to report the organs that chitosan acted on, and gene expression profiles of chitosan-targeted organs were further analyzed to elucidate the mechanisms of chitosan. Bioluminescent imaging showed that constitutive PPAR activities were detected in brain and gastrointestinal tract. Administration of chitosan significantly activated the PPAR activities in brain and stomach. Microarray analysis of brain and stomach showed that several pathways involved in lipid and glucose metabolism were regulated by chitosan. Moreover, the expression levels of metabolism-associated genes like apolipoprotein B (apoB) and ghrelin genes were down-regulated by chitosan. In conclusion, these findings suggested the feasibility of PPAR bioluminescent imaging-guided transcriptomic analysis on the evaluation of chitosan-affected metabolic responses in vivo. Moreover, we newly identified that downregulated expression of apoB and ghrelin genes were novel mechanisms for chitosan-affected metabolic responses in vivo. PMID:22496881

In Vivo Bioluminescence Tomography for Monitoring Breast Tumor Growth and Metastatic Spreading: Comparative Study and Mathematical Modeling

PubMed Central

Mollard, Séverine; Fanciullino, Raphaelle; Giacometti, Sarah; Serdjebi, Cindy; Benzekry, Sebastien; Ciccolini, Joseph

2016-01-01

This study aimed at evaluating the reliability and precision of Diffuse Luminescent Imaging Tomography (DLIT) for monitoring primary tumor and metastatic spreading in breast cancer mice, and to develop a biomathematical model to describe the collected data. Using orthotopic mammary fat pad model of breast cancer (MDAMB231-Luc) in mice, we monitored tumor and metastatic spreading by three-dimensional (3D) bioluminescence and cross-validated it with standard bioluminescence imaging, caliper measurement and necropsy examination. DLIT imaging proved to be reproducible and reliable throughout time. It was possible to discriminate secondary lesions from the main breast cancer, without removing the primary tumor. Preferential metastatic sites were lungs, peritoneum and lymph nodes. Necropsy examinations confirmed DLIT measurements. Marked differences in growth profiles were observed, with an overestimation of the exponential phase when using a caliper as compared with bioluminescence. Our mathematical model taking into account the balance between living and necrotic cells proved to be able to reproduce the experimental data obtained with a caliper or DLIT imaging, because it could discriminate proliferative living cells from a more composite mass consisting of tumor cells, necrotic cell, or inflammatory tissues. DLIT imaging combined with mathematical modeling could be a powerful and informative tool in experimental oncology. PMID:27812027

Transient transfection of mammalian cells using a violet diode laser

NASA Astrophysics Data System (ADS)

Torres-Mapa, Maria Leilani; Angus, Liselotte; Ploschner, Martin; Dholakia, Kishan; Gunn-Moore, Frank J.

2010-07-01

We demonstrate the first use of the violet diode laser for transient mammalian cell transfection. In contrast to previous studies, which showed the generation of stable cell lines over a few weeks, we develop a methodology to transiently transfect cells with an efficiency of up to ~40%. Chinese hamster ovary (CHO-K1) and human embryonic kidney (HEK293) cells are exposed to a tightly focused 405-nm laser in the presence of plasmid DNA encoding for a mitochondrial targeted red fluorescent protein. We report transfection efficiencies as a function of laser power and exposure time for our system. We also show, for the first time, that a continuous wave laser source can be successfully applied to selective gene silencing experiments using small interfering RNA. This work is a major step towards an inexpensive and portable phototransfection system.

Minimally-Invasive Gene Transfection by Chemical and Physical Interaction of Atmospheric Pressure Plasma Flow

NASA Astrophysics Data System (ADS)

Kaneko, Toshiro

2014-10-01

Non-equilibrium atmospheric pressure plasma irradiated to the living-cell is investigated for medical applications such as gene transfection, which is expected to play an important role in molecular biology, gene therapy, and creation of induced pluripotent stem (iPS) cells. However, the conventional gene transfection using the plasma has some problems that the cell viability is low and the genes cannot be transferred into some specific lipid cells, which is attributed to the unknown mechanism of the gene transfection using the plasma. Therefore, the time-controlled atmospheric pressure plasma flow is generated and irradiated to the living-cell suspended solution for clarifying the transfection mechanism toward developing highly-efficient and minimally- invasive gene transfection system. In this experiment, fluorescent dye YOYO-1 is used as the simulated gene and LIVE/DEAD Stain is simultaneously used for cell viability assay. By the fluorescence image, the transfection efficiency is calculated as the ratio of the number of transferred and surviving cells to total cell count. It is clarified that the transfection efficiency is significantly increased by the short-time (<4 sec) and short-distance (<40 mm) plasma irradiation, and the high transfection efficiency of 53% is realized together with the high cell viability (>90%). This result indicates that the physical effects such as the electric field caused by the charged particles arriving at the surface of the cell membrane, and chemical effects associated with plasma-activated products in solution act synergistically to enhance the cell-membrane transport with low-damage. This work was supported by JSPS KAKENHI Grant Number 24108004.

Monitoring tumor metastases and osteolytic lesions with bioluminescence and micro CT imaging.

PubMed

Lim, Ed; Modi, Kshitij; Christensen, Anna; Meganck, Jeff; Oldfield, Stephen; Zhang, Ning

2011-04-14

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location. Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research.

PubMed

Siciliano, Giulia; Alano, Pietro

2015-01-01

The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have been essential to unveil mechanisms of parasite gene expression and to develop in vivo imaging approaches in mouse malaria models. Mainly the human malaria parasite Plasmodium falciparum and the rodent parasite P. berghei have been engineered to express bioluminescent reporters in almost all the developmental stages of the parasite along its complex life cycle between the insect and the vertebrate hosts. Plasmodium lines expressing conventional and improved luciferase reporters are now gaining a central role to develop cell based assays in the much needed search of new antimalarial drugs and to open innovative approaches for both fundamental and applied research in malaria.

A novel rapid and reproducible flow cytometric method for optimization of transfection efficiency in cells

PubMed Central

Homann, Stefanie; Hofmann, Christian; Gorin, Aleksandr M.; Nguyen, Huy Cong Xuan; Huynh, Diana; Hamid, Phillip; Maithel, Neil; Yacoubian, Vahe; Mu, Wenli; Kossyvakis, Athanasios; Sen Roy, Shubhendu; Yang, Otto Orlean

2017-01-01

Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness. PMID:28863132

Carbon nanoparticles for gene transfection in eukaryotic cell lines.

PubMed

Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

2014-06-01

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests. Copyright © 2014 Elsevier B.V. All rights reserved.

Enhanced Beetle Luciferase for High-Resolution Bioluminescence Imaging

PubMed Central

Nakajima, Yoshihiro; Yamazaki, Tomomi; Nishii, Shigeaki; Noguchi, Takako; Hoshino, Hideto; Niwa, Kazuki; Viviani, Vadim R.; Ohmiya, Yoshihiro

2010-01-01

We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin α by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc. PMID:20368807

Inorganic nanoparticles for transfection of mammalian cells and removal of viruses from aqueous solutions.

PubMed

Link, Nils; Brunner, Tobias J; Dreesen, Imke A J; Stark, Wendelin J; Fussenegger, Martin

2007-12-01

Owing to their small size, synthetic nanoparticles show unprecedented biophysical and biochemical properties which may foster novel advances in life-science research. Using flame-spray synthesis technology we have produced non-coated aluminum-, calcium-, cerium-, and zirconium-derived inorganic metal oxide nanoparticles which not only exhibit high affinity for nucleic acids, but can sequester such compounds from aqueous solution. This non-covalent DNA-binding capacity was successfully used to transiently transfect a variety of mammalian cells including human, reaching transfection efficiencies which compared favorably with classic calcium phosphate precipitation (CaP) procedures and lipofection. In this straightforward protocol, transfection was enabled by simply mixing nanoparticles with DNA in solution prior to addition to the target cell population. Transiently transfected cells showed higher production levels of the human secreted glycoprotein SEAP compared to isogenic populations transfected with established technologies. Inorganic metal oxide nanoparticles also showed a high binding capacity to human-pathogenic viruses including adenovirus, adeno-associated virus and human immunodeficiency virus type 1 and were able to clear these pathogens from aqueous solutions. The DNA transfection and viral clearance capacities of inorganic metal oxide nanoparticles may provide cost-effective biopharmaceutical manufacturing and water treatment in developing countries.

Intracellular pathways and nuclear localization signal peptide-mediated gene transfection by cationic polymeric nanovectors.

PubMed

Hu, Qinglian; Wang, Jinlei; Shen, Jie; Liu, Min; Jin, Xue; Tang, Guping; Chu, Paul K

2012-02-01

Polyethylenimine (PEI) - based polymers are promising cationic nanovectors. A good understanding of the mechanism by which cationic polymers/DNA complexes are internalized and delivered to nuclei helps to identify which transport steps may be manipulated in order to improve the transfection efficiency. In this work, cell internalization and trafficking of PEI-CyD (PC) composed of β-cyclodextrin (β-CyD) and polyethylenimine (PEI, Mw 600) are studied. The results show that the PC transfected DNA is internalized by binding membrane-associated proteoglycans. The endocytic pathway of the PC particles is caveolae- and clathrin-dependent with both pathways converging to the lysosome. The intracellular fate of the PC provides visual evidence that it can escape from the lysosome. Lysosomal inhibition with chloroquine has no effect on PC mediated transfection implying that blocking the lysosomal traffic does not improve transfection. To improve the nuclear delivery of PC transfected DNA, nuclear localization signal (NLS) peptides are chosen to conjugate and combine with the PC. Compared to PC/pDNA, PC-NLS/pDNA, and PC/pDNA/NLS can effectively improve gene transfection in dividing and non-dividing cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Ultrasonic destruction of albumin microbubbles enhances gene transfection and expression in cardiac myocytes.

PubMed

Wang, Guo-zhong; Liu, Jing-hua; Lü, Shu-zheng; Lü, Yun; Guo, Cheng-jun; Zhao, Dong-hui; Fang, Dong-ping; He, Dong-fang; Zhou, Yuan; Ge, Chang-jiang

2011-05-01

It has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes. The β-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed. The ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P < 0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced β-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05). Ultrasonic

Development of a chemiluminescent and bioluminescent system for the detection of bacteria in wastewater effluent

NASA Technical Reports Server (NTRS)

Thomas, R. R.

1975-01-01

Automated chemiluminescent and bioluminescent sensors were developed for continuous monitoring of microbial levels in wastewater effluent. Development of the chemiluminescent system included optimization of reagent concentrations as well as two new techniques which will allow for increased sensitivity and specificity. The optimal reagent concentrations are 0.0025 M luminol and 0.0125 M sodium perborate in 0.75N sodium hydroxide before addition of sample. The methods developed to increase specificity include (1) extraction of porphyrins from bacteria collected in a filter using 0.1N NaOH - 50 percent Ethanol, and (2) use of the specific reaction rate characteristics for the different luminol catalysts. Since reaction times are different for each catalyst, the reaction can be made specific for bacteria by measuring only the light emission from the particular reaction time zone specific for bacteria. Developments of the bioluminescent firefly luciferase system were in the area of flow system design.

Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin-luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin.

PubMed

Furuya, Takahito; Takehara, Issey; Shimura, Asuka; Kishimoto, Hisanao; Yasujima, Tomoya; Ohta, Kinya; Shirasaka, Yoshiyuki; Yuasa, Hiroaki; Inoue, Katsuhisa

2018-01-15

Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (K m ) of 0.23 μM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent K m of 0.36 μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells. Copyright © 2017 Elsevier Inc. All rights reserved.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines.

PubMed

Regla-Nava, Jose A; Jimenez-Guardeño, Jose M; Nieto-Torres, Jose L; Gallagher, Thomas M; Enjuanes, Luis; DeDiego, Marta L

2013-11-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Antitumoral effect of IL-12 gene transfected via liposomes into B16F0 cells.

PubMed

Speroni, Lucía; Gasparri, Julieta; de los A Bustuoabad, Victoria; Chiaramoni, Nadia S; Smagur, Andrzej; Szala, Stanisław; Taira, María C; del V Alonso, Silvia

2009-01-01

Murine melanoma B16F0 cells were transfected with SA:DPPC:DOPE (2:1:1 molar ratio) liposomes associated with a plasmid encoding murine IL-12. Stearylamine, a cationic lipid, showed a greater transfection efficiency compared to DOTAP-containing liposomes. The lipid:DNA ratio was 2:1 (w/w). Control groups were mock transfected or transfected with an empty plasmid (pNeo). pNeo or IL-12 transfected cells and controls were inoculated intradermically into the dorsal region of the foot or the lateral flank of C57BL6 mice. Results showed that IL-12 expression had a marked effect on in vivo growth of B16 melanoma tumors developed in both anatomic sites, significantly retarding their growth and prolonging host survival.

Bioluminescent bioreporter integrated circuit devices and methods for detecting ammonia

DOEpatents

Simpson, Michael L [Knoxville, TN; Paulus, Michael J [Knoxville, TN; Sayler, Gary S [Blaine, TN; Applegate, Bruce M [West Lafayette, IN; Ripp, Steven A [Knoxville, TN

2007-04-24

Monolithic bioelectronic devices for the detection of ammonia includes a microorganism that metabolizes ammonia and which harbors a lux gene fused with a heterologous promoter gene stably incorporated into the chromosome of the microorganism and an Optical Application Specific Integrated Circuit (OASIC). The microorganism is generally a bacterium.

Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

NASA Astrophysics Data System (ADS)

Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

2016-04-01

Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes.

PubMed

Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A; Cheng, Shuk Han; Sun, Dong

2016-04-12

Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

An Assessment and Annotated Bibliography of Marine Bioluminescence Research: 1979-1987.

DTIC Science & Technology

1993-01-01

to Leona Cole, Division Secretary for NOARL Code 330, who prepared the typed draft of this manuscript. The mention of commercial products or company...look at the end points of the curves suggest a The graph shows the number of publications each small drop in research productivity in three areas; 0 year...Table 2 shows the most productive agencies dominated the funding for U.S. research institutions. In the next decade, we may expect on bioluminescence

Simulation of micro/nano electroporation for cell transfection

NASA Astrophysics Data System (ADS)

Zhang, Guocheng; Fan, Na; Jiang, Hai; Guo, Jian; Peng, Bei

2018-03-01

The 3D micro/nano electroporation for transfection has become a powerful biological cell research technique with the development of micro-nano manufacturing technology. The micro channels connected the cells with transfection reagents on the chip were important to the transmemnbrane potentical, which directly influences the electroporation efficiency. In this study, a two-dimensional model for electroporation of cells was designed to address the effects of channels’ sizes and number on transmembrane potential. The simulation results indicated that the transmembrane potential increased with increasing size of channels’ entrances. Moreover, compared with single channel entrance, the transmembrane potential was higher when the cells located at multiple channels entrances. These results suggest that it IS required to develop higher micro manufacturing technology to create channels as we expected size.

Using a nanopore for single molecule detection and single cell transfection.

PubMed

Nelson, Edward M; Kurz, Volker; Shim, Jiwook; Timp, Winston; Timp, Gregory

2012-07-07

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.

Properties of the Deep Scattering Layer Analyzed in Terms of Bioluminescent Behavior of Its Components.

DTIC Science & Technology

1984-02-28

15:203-221. 1975 Anderson, P. & Case, J.F. Electrical activity associated with luminescence and other colonial behavior in the pennatulid Renilla ...Development of bioluminescence and other effector responses in the pennatulid coelentrate Renilla kollikeri. Biol. Bull. 157:506-523. Satterlie, R.A. & Case

In vitro and in vivo transfection of primary phagocytes via microbubble-mediated intraphagosomal sonoporation.

PubMed

Lemmon, Jason C M; McFarland, Ryan J; Rybicka, Joanna M; Balce, Dale R; McKeown, Kyle R; Krohn, Regina M; Matsunaga, Terry O; Yates, Robin M

2011-08-31

The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalized lipid-based microbubbles. This approach enables the transfection of primary phagocytes in vitro, with a modest, but reliable efficiency. Furthermore, this methodology was readily adapted to transfect murine peritoneal macrophages in vivo. This technology has immediate application to current research efforts and has potential for use in gene therapy and vaccination strategies. Copyright © 2011 Elsevier B.V. All rights reserved.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

PubMed

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

PubMed

Wu, Qing-Ping; Zhang, Lei; Shao, Xiao-Xia; Wang, Jia-Hui; Gao, Yu; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

2016-04-01

Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1.

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

PubMed Central

Heinemann, Dag; Schomaker, Markus; Kalies, Stefan; Schieck, Maximilian; Carlson, Regina; Escobar, Hugo Murua; Ripken, Tammo; Meyer, Heiko; Heisterkamp, Alexander

2013-01-01

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types. PMID:23536802

Structure relationship of cationic lipids on gene transfection mediated by cationic liposomes.

PubMed

Paecharoenchai, Orapan; Niyomtham, Nattisa; Apirakaramwong, Auayporn; Ngawhirunpat, Tanasait; Rojanarata, Theerasak; Yingyongnarongkul, Boon-ek; Opanasopit, Praneet

2012-12-01

The aim of this study was to investigate the transfection efficiency of cationic liposomes formulated with phosphatidylcholine (PC) and novel synthesized diethanolamine-based cationic lipids at a molar ratio of 5:1 in comparison with Lipofectamine™ 2000. Factors affecting transfection efficiency and cell viability, including the chemical structure of the cationic lipids, such as different amine head group (diamine and polyamine; and non-spermine and spermine) and acyl chain lengths (C14, C16, and C18) and the weight ratio of liposomes to DNA were evaluated on a human cervical carcinoma cell line (HeLa cells) using the pDNA encoding green fluorescent protein (pEGFP-C2). Characterizations of these lipoplexes in terms of size and charge measurement and agarose gel electrophoresis were performed. The results from this study revealed that almost no transfection was observed in the liposome formulations composed of cationic lipids with a non-spermine head group. In addition, the transfection efficiency of these cationic liposomes was in the following order: spermine-C14 > spermine-C16 > spermine-C18. The highest transfection efficiency was observed in the formulation of spermine-C14 liposomes at a weight ratio of 25; furthermore, this formulation was safe for use in vitro. In conclusion, cationic liposomes containing spermine head groups demonstrated promising potential as gene carriers.

[Study of the antitumor activity of alveolar macrophages after transfected human INF-γ gene].

PubMed

Zhou, Fengli; Bi, Xiaogang; Zhang, Tiantuo; Huang, Jing

2011-05-01

Alveolar macrophages (AMs) activated have the antitumor activity. The interferon-γ (INF-γ) is one of the stimulating factors. INF-γ can enhance the immune function of AMs in vitro. The aim of this study is to investigate the effect of human INF-γ gene on the antitumor activity of AMs when transfected into the alveolar macrophages (AMs) from the patients with lung cancer in vitro. AMs obtained by brochoalveolar lavage were separated and transfected by INF-γ gene. RT-PCR and ELISA were applied to determine whether the transfection was successful. The levels of tumor necrosis factor α (TNF-α), nitric oxide (NO) and interleukin-1 (IL-1) produced by AMs and the killing activity of AMs against L1210 cells was detected respectively. Both RT-PCR and ELISA demonstrated that human INF-γ gene had been successfully transfected into AMs. When transfected by human INF-γ gene, the levels of TNF-α, NO and IL-1 produced by AMs from the patients with lung cancer and the killing activity of AMs against L1210 cells were significantly higher than those of the control groups. Human INF-γ gene can enhance the antitumor activity of AMs when transfected into AMs from the patients with lung cancer.

[Construction of the superantigen SEA transfected laryngocarcinoma cells].

PubMed

Ji, Xiaobin; Jingli, J V; Liu, Qicai; Xie, Jinghua

2013-04-01

To construct an eukaryotic expression vectors containing superantigen staphylococcal enterotoxin A (SEA) gene, and to identify its expression in laryngeal squamous carcinoma cells. SEA full-length gene fragment was obtained from ATCC13565 genome of the staphylococcus, referencing standard strains producing SEA. Coding sequence of SEA was artificially synthetized. Than, SEA gene fragments was subcloned into eukaryotic expression vector pIRES-EGFP. The recombinant plasmid pSEA-IRES-EGFP was constructed and was transfected to laryngocarcinoma Hep-2 cells. Resistant clones were screened by G418. The expression of SEA in laryngocarcinoma cells was identified with ELISA and RT-PCR method. The subclone of artificially synthetized SEA gene was subclone to eukaryotic expression vector pires-EGFP. Flanking sequence confirmed that SEA sequence was fully identical to the coding sequence of standard staphylococcus strains ATCC13565 in Genbank. After recombinant plasmid transfected to laryngocarcinoma cells, the resistant clones was obtained after screening for two weeks. The clones were selected. The specific gene fragment was obtained by RT-PCR amplification. ELISA assay confirmed that the content of SEA protein in supernatant fluid of cell culture had reached about Pg level. The recombinant eukaryotic expression vector containing superantigen SEA gene is successfully constructed, and is capable of effective expression and continued secretion of SEA protein in laryngochrcinoma Hep-2 cells after recombinant plasmid transfected to laryngocarcinoma cells.

Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide

PubMed Central

Vellani, Vittorio; Mapplebeck, Sarah; Moriondo, Andrea; Davis, John B; McNaughton, Peter A

2001-01-01

The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. Removing external Ca2+ enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium

Firefly luciferase-based dynamic bioluminescence imaging: a noninvasive technique to assess tumor angiogenesis.

PubMed

Sun, Amy; Hou, Lewis; Prugpichailers, Tiffany; Dunkel, Jason; Kalani, Maziyar A; Chen, Xiaoyuan; Kalani, M Yashar S; Tse, Victor

2010-04-01

Bioluminescence imaging (BLI) is emerging as a cost-effective, high-throughput, noninvasive, and sensitive imaging modality to monitor cell growth and trafficking. We describe the use of dynamic BLI as a noninvasive method of assessing vessel permeability during brain tumor growth. With the use of stereotactic technique, 10 firefly luciferase-transfected GL26 mouse glioblastoma multiforme cells were injected into the brains of C57BL/6 mice (n = 80). After intraperitoneal injection of D-luciferin (150 mg/kg), serial dynamic BLI was performed at 1-minute intervals (30 seconds exposure) every 2 to 3 days until death of the animals. The maximum intensity was used as an indirect measurement of tumor growth. The adjusted slope of initial intensity (I90/Im) was used as a proxy to monitor the flow rate of blood into the vascular tree. Using a modified Evans blue perfusion protocol, we calculated the relative permeability of the vascular tree at various time points. Daily maximum intensity correlated strongly with tumor volume. At postinjection day 23, histology and BLI demonstrated an exponential growth of the tumor mass. Slopes were calculated to reflect the flow in the vessels feeding the tumor (adjusted slope = I90/Im). The increase in BLI intensity was correlated with a decrease in adjusted slope, reflecting a decrease in the rate of blood flow as tumor volume increased (y = 93.8e-0.49, R2 = 0.63). Examination of calculated slopes revealed a peak in permeability around postinjection day 20 (n = 42, P < .02 by 1-way analysis of variance) and showed a downward trend in relation to both postinjection day and maximum intensity observed; as angiogenesis progressed, tumor vessel caliber increased dramatically, resulting in sluggish but increased flow. This trend was correlated with Evans blue histology, revealing an increase in Evans blue dye uptake into the tumor, as slope calculated by BLI increases. Dynamic BLI is a practical, noninvasive technique that can

Bioluminescence in a complex coastal environment: 1. Temporal dynamics of nighttime water-leaving radiance

NASA Astrophysics Data System (ADS)

Moline, Mark A.; Oliver, Matthew J.; Mobley, Curtis D.; Sundman, Lydia; Bensky, Thomas; Bergmann, Trisha; Bissett, W. Paul; Case, James; Raymond, Erika H.; Schofield, Oscar M. E.

2007-11-01

Nighttime water-leaving radiance is a function of the depth-dependent distribution of both the in situ bioluminescence emissions and the absorption and scattering properties of the water. The vertical distributions of these parameters were used as inputs for a modified one-dimensional radiative transfer model to solve for spectral bioluminescence water-leaving radiance from prescribed depths of the water column. Variation in the water-leaving radiance was consistent with local episodic physical forcing events, with tidal forcing, terrestrial runoff, particulate accumulation, and biological responses influencing the shorter timescale dynamics. There was a >90 nm shift in the peak water-leaving radiance from blue (˜474 nm) to green as light propagated to the surface. In addition to clues in ecosystem responses to physical forcing, the temporal dynamics in intensity and spectral quality of water-leaving radiance provide suitable ranges for assessing detection. This may provide the information needed to estimate the depth of internal light sources in the ocean, which is discussed in part 2 of this paper.

[Experimental study on dog's bone marrow stem cells transfected by pIRES2-EGFP-IGF-1 gene].

PubMed

Zhu, Guo-qiang; Wu, Zhi-fen; Li, Yuan-fei; Hu, De-hua; Wang, Qin-tao

2006-12-01

To establish the bone marrow stem cells (MSC) model which could highly express the insulin-like growth factor 1 (IGF-1) transfected by dog's IGF-1 gene. pIRES2-EGFP-IGF-1 was transfected into MSC by lipofectamine. Positive clones were selected with G418. The expression of IGF-1 protein in the MSC was determined by immunohistochemistry and Western blot analysis. The IGF-1 in the supernatant of the transfected MSC was detected by sandwich-in ELISA. The periodontal ligament cells (PDLC) were cultured in the supernatant of the transfected MSC. The changes of PDLC' proliferation were observed by MTT. IGF-1-transfected MSC could apparently express IGF-1. The IGF-1 protein in the supernatant of the transfected MSC was confirmed by sandwich-in ELISA. IGF-1 could promote the PDLC' proliferation. The MSC transfected by dog's IGF-1 gene can highly express IGF-1, which may lay the foundation for further study on periodontal regeneration.

Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.

PubMed Central

McPhaul, M; Berg, P

1986-01-01

The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to produce detectable ASGP-R. The affinity of transduced ASGP-R for asialo orosomucoid is less than that of the native rat ASGP-R, and the number of surface receptors in clones expressing ASGP-R is about one-fifth that found on rat hepatocytes. Images PMID:3466162

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

PubMed Central

1993-01-01

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC

Identification of a vacuolar proton channel that triggers the bioluminescent flash in dinoflagellates.

PubMed

Rodriguez, Juan D; Haq, Saddef; Bachvaroff, Tsvetan; Nowak, Kristine F; Nowak, Scott J; Morgan, Deri; Cherny, Vladimir V; Sapp, Maredith M; Bernstein, Steven; Bolt, Andrew; DeCoursey, Thomas E; Place, Allen R; Smith, Susan M E

2017-01-01

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel (HV1) that opens in response to depolarizing voltage across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thereby causing a pH drop that triggers light emission. HV1 channels were subsequently identified and demonstrated to have important functions in a multitude of eukaryotic cells. Here we report a predicted protein from Lingulodinium polyedrum that displays hallmark properties of bona fide HV1, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. Western blotting and fluorescence confocal microscopy of isolated L. polyedrum scintillons immunostained with antibody to LpHV1 confirm LpHV1's predicted organellar location. Proteomics analysis demonstrates that isolated scintillon preparations contain peptides that map to LpHV1. Finally, Zn2+ inhibits both LpHV1 proton current and the acid-induced flash in isolated scintillons. These results implicate LpHV1 as the voltage gated proton channel that triggers bioluminescence in L. polyedrum, confirming Hastings' hypothesis. The same channel likely mediates the action potential that communicates the signal along the tonoplast to the scintillon.

[Optimizing condition for oligofectamine-mediated SP1 decoy oligodeoxynucleotides transfection into SV-40-PED cells].

PubMed

Huang, Yabing; Xu, Jing; Chen, Song

2007-08-01

To determine the optimizing parameters in transfecting the SV-40-PED cells mediated by oligofectamine. With a change of Decoy oligodeoxynucleotides (ODNs)/oligofectamine in ratio and the transfection time, the uptake rate and the mean fluorescence intensity of SP1 ODNs in the SV-40-PED cells were measured by flow cytometry to evaluate the transfection efficiencies. 4 microl oligofectamine with different concentrations of ODNs(2.5, 5.0, 7.5, 10.0 and 12.5 microl) were put into 100 microl of DMEM without serum and antibiotics. the (SV-40-PED) cells were transfected after 20 min at room temperature. the final concentration of SP1 decay ODNs were 50,100, 150, 200 and 250 nmol/L. Transfection effieiency was detected at 26 h after transfection. The intracellular distribution of SP1 ODNs was determined with a fluorescence microscope. The lactate dehydrogenase (LDH) activity in the supernatant was measured to assess the cytotoxicity. The uptake of SP1 ODNs into the SV-40-PED cells was significantly improved by oligofectamine. The cell appearance did not change much in the groups of 50, 100 and 150 nmol/L. In the groups of 200 and 250 nmol/L, the cell reverted after being shrinked and altered to round. At 26 h after the transfection, there was no marked change in the cell form at the concentration of 250 nmol/L. There was floatation at 48 and 72 h after the transfection. Under the fluorescence microscope, we observed fluorescent materials distributed in the cell nucleus in the successfully-transferred groups. We could see the nucleoli clearly in the groups of 200 nmol/L and 250 nmol/L. There was a stronger fluorescence intensity with a higher concentration and the fluorescent materials gathered at the cell nucleus. At the final concentration of 250 nmol/L, the LDH level was 137.12+/-3.92 U/L in the 72-h group, which was significantly higher those that in the 26-h group (49.61+/-17.13 U/L) and the 48-h group (120.26 +/- 8.42 U/L) (P<0.01). At 26 h after the transfection

The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha1A1-adrenoceptors.

PubMed

Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork; Pickering, Darryl S; Kristensen, Torsten

2008-10-31

Antipsychotic drugs often cause orthostatic hypotension, probably through antagonist action on resistance vessel alpha(1A)-adrenoceptors. Here we have tested this possibility directly using cells transfected with a relevant human alpha(1A)-adrenoceptor splice variant. To determine a splice variant which was relevant, we used quantitative real-time polymerase chain reaction (qPCR) to determine the prevalence in human subcutaneous small arteries of three of the five splice variants ADRA1A_v1-5, which encode functional protein: alpha(1A1)-, alpha(1A3)-, alpha(1A4)-adrenoceptors. Our statistical analysis showed higher transcription levels of alpha(1A1)- than of alpha(1A3)- and alpha(1A4)-adrenoceptors (1.6 and 5.8 times, respectively). We therefore chose to study the alpha(1A1)-adrenoceptor, and the cDNA encoding it was transfected into the Flp-In-293 (modified from HEK-293) cell line to produce a cell line stably expressing a functional form of this splice variant. The expression of recombinant alpha(1A1)-adrenoceptor subtype was confirmed by Western immunoblot analysis, and its functionality demonstrated using a Fura-2 assay by a rise in intracellular calcium concentration ([Ca(2+)](i)) when challenged with phenylephrine (EC(50)=1.61x10(-8) M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration-response curves for phenylephrine in cells expressing human recombinant alpha(1A1)-adrenoceptors to yield pK(B) values of 8.40, 8.05, 8.26 and 7.38, respectively. In [7-methoxy-(3)H]-prazosin binding experiments, high expression was seen (B(max)=48.5+/-16.7 pmol/mg protein, +/-S.E.M.) along with high affinity binding to a single site (K(d)=0.210+/-0.034 nM). The pharmacological profiles of recombinant human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In

Modular low-light microscope for imaging cellular bioluminescence and radioluminescence

PubMed Central

Kim, Tae Jin; Türkcan, Silvan; Pratx, Guillem

2017-01-01

Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy, such as bioluminescence, chemiluminescence, or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back-to-back from each other, using standard optomechanical components. We also provide directions on how to image dim signals such as radioluminescence (1-1.5 h), bioluminescence (∼30 min) and low-excitation fluorescence (∼15 min). In particular, radioluminescence microscopy is explained in detail as it is a newly developed technique, which enables the study of small molecule transport (eg. radiolabeled drugs, metabolic precursors, and nuclear medicine contrast agents) by single cells without perturbing endogenous biochemical processes. In this imaging technique, a scintillator crystal (eg. CdWO4) is placed in close proximity to the radiolabeled cells, where it converts the radioactive decays into optical flashes detectable using a sensitive camera. Using the image reconstruction toolkit provided in this protocol, the flashes can be reconstructed to yield high-resolution image of the radiotracer distribution. With appropriate timing, the three aforementioned imaging modalities may be performed altogether on a population of live cells, allowing the user to perform parallel functional studies of cell heterogeneity at the single-cell level. PMID:28426025

Evaluation of cationic liposomes composed of an amino acid-based lipid for neuronal transfection.

PubMed

Obata, Yosuke; Ciofani, Gianni; Raffa, Vittoria; Cuschieri, Alfred; Menciassi, Arianna; Dario, Paolo; Takeoka, Shinji

2010-02-01

We investigated the ability of cationic liposomes composed of 1,5-dihexadecyl N-arginyl-L-glutamate (Arg-Glu2C(16)) to carry nucleic acids into neuronal cells. Such liposomes have been shown to have a remarkable capacity for transfecting immortalized cell lines. Lipoplexes between the Arg-Glu2C(16) liposomes and plasmid DNA encoding green fluorescent protein (GFP) were analyzed in terms of lipoplex formation, intracellular DNA trafficking, transfection efficiency, and cytotoxicity in neuronal SH-SY5Y cells. A maximum number of cells expressing GFP was obtained with lipoplexes at a lipid-to-DNA ratio of 15. With these lipoplexes, 16% of the cells were GFP-positive, which was approximately fourfold higher than the level obtained with a commercially available transfection reagent, Lipofectamine 2000. Furthermore, as a result of the low cytotoxicity of the Arg-Glu2C(16) lipoplexes, the proportion of GFP-positive cells could be increased to 25% by increasing the concentration of lipoplexes that was applied to the cells. We have demonstrated that Arg-Glu2C(16), as a model cationic amino acid-based lipid, has a high capability as a gene carrier, even for neuronal transfection. In this study, specific cationic liposomes were characterized as nucleic acid transfection agents for neuronal cells. A fourfold higher transfection rate with low cytotoxicity was reported compared to Lipofectamine 2000, a commercial reagent. The authors conclude that the studied cationic liposomes have a high capability as a gene carrier for neuronal transfection. This may become clinically significant in future gene therapy efforts of neuronal diseases. Copyright 2010 Elsevier Inc. All rights reserved.

Quantum/molecular mechanics study of firefly bioluminescence on luciferase oxidative conformation

NASA Astrophysics Data System (ADS)

Pinto da Silva, Luís; Esteves da Silva, Joaquim C. G.

2014-07-01

This is the first report of a computational study of the color tuning mechanism of firefly bioluminescence, using the oxidative conformation of luciferase. The results of these calculations demonstrated that the electrostatic field generated by luciferase is fundamental both for the emission shift and efficiency. Further calculations indicated that a shift in emission is achieved by modulating the energy, at different degrees, of the emissive and ground states. These differences in energy modulation will then lead to changes in the energy gap between the states.

Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

PubMed

Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

2015-08-07

Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly

Synergistic effect of electrical and chemical factors on endocytosis in micro-discharge plasma gene transfection

NASA Astrophysics Data System (ADS)

Jinno, M.; Ikeda, Y.; Motomura, H.; Isozaki, Y.; Kido, Y.; Satoh, S.

2017-06-01

We have developed a new micro-discharge plasma (MDP)-based gene transfection method, which transfers genes into cells with high efficiency and low cytotoxicity; however, the mechanism underlying the method is still unknown. Studies revealed that the N-acetylcysteine-mediated inhibition of reactive oxygen species (ROS) activity completely abolished gene transfer. In this study, we used laser-produced plasma to demonstrate that gene transfer does not occur in the absence of electrical factors. Our results show that both electrical and chemical factors are necessary for gene transfer inside cells by microplasma irradiation. This indicates that plasma-mediated gene transfection utilizes the synergy between electrical and chemical factors. The electric field threshold required for transfection was approximately 1 kV m-1 in our MDP system. This indicates that MDP irradiation supplies sufficient concentrations of ROS, and the stimulation intensity of the electric field determines the transfection efficiency in our system. Gene transfer by plasma irradiation depends mainly on endocytosis, which accounts for at least 80% of the transfer, and clathrin-mediated endocytosis is a dominant endocytosis. In plasma-mediated gene transfection, alterations in electrical and chemical factors can independently regulate plasmid DNA adhesion and triggering of endocytosis, respectively. This implies that plasma characteristics can be adjusted according to target cell requirements, and the transfection process can be optimized with minimum damage to cells and maximum efficiency. This may explain how MDP simultaneously achieves high transfection efficiency with minimal cell damage.

Mechanisms of Nanoparticle Mediated siRNA Transfection by Melittin-Derived Peptides

PubMed Central

Hou, Kirk K.; Pan, Hua; Ratner, Lee; Schlesinger, Paul H.; Wickline, Samuel A.

2014-01-01

Traditional peptide-mediated siRNA transfection via peptide transduction domains exhibits limited cytoplasmic delivery of siRNA due to endosomal entrapment. This work overcomes these limitations with the use of membrane-destabilizing peptides derived from melittin for the knockdown of NFkB signaling in a model of adult T-Cell leukemia/lymphoma. While the mechanism of siRNA delivery into the cytoplasmic compartment by peptide transduction domains has not been well studied, our analysis of melittin derivatives indicates that concurrent nanocomplex disassembly and peptide-mediated endosomolysis are crucial to siRNA transfection. Importantly, in the case of the most active derivative, p5RHH, this process is initiated by acidic pH, indicating that endosomal acidification after macropinocytosis can trigger siRNA release into the cytoplasm. These data provide general principles regarding nanocomplex response to endocytosis which may guide the development of peptide/siRNA nanocomplex-based transfection. PMID:24053333

Evaluation of a bioluminescent mouse model expressing aromatase PII-promoter-controlled luciferase as a tool for the study of endocrine disrupting chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivest, Patricia, E-mail: patricia.rivest@iaf.inrs.ca; Devine, Patrick J., E-mail: patrick.devine@iaf.inrs.ca; Sanderson, J. Thomas, E-mail: thomas.sanderson@iaf.inrs.c

Dysfunction of the enzyme aromatase (CYP19) is associated with endocrine pathologies such as osteoporosis, impaired fertility and development of hormone-dependent cancers. Certain endocrine disrupting chemicals affect aromatase expression and activity in vitro, but little is known about their ability to do so in vivo. We evaluated a bioluminescent mouse model (LPTA (registered)) CD-1-Tg(Cyp19-luc)-Xen) expressing luciferase under control of the gonadal aromatase pII promoter as an in vivo screening tool for chemicals that may affect aromatase expression. We studied the effects of forskolin, pregnant mare serum gonadotropin and atrazine in this model (atrazine was previously shown to induced pII-promoter-driven aromatase expressionmore » in H295R human adrenocortical carcinoma cells). About 2-4 out of every group of 10 male or female Cyp19-luc mice injected i.p. with 10 mg/kg forskolin had increased gonadal bioluminescence after 3-5 days compared to controls; the others appeared non-responsive. Similarly, about 4 per group of 9 individual females injected with pregnant mare serum gonadotropin had increased ovarian bioluminescence after 24 h. There was a statistically significant correlation between ovarian bioluminescence and plasma estradiol concentrations (n = 14; p = 0.022). Males exposed to a single dose of 100 mg/kg or males and females exposed to 5 daily injections of 30 mg/kg atrazine showed no change in gonadal bioluminescence over a 7 day period, but a significant interaction was found between atrazine (100 mg/kg) and time in female mice (p < 0.05; two-way ANOVA). Ex vivo luciferase activity in dissected organs was increased by forskolin in testis, epididymis and ovaries. Atrazine (30 mg/kg/day) increased (30%) luciferase activity significantly in epididymis only. In conclusion, certain individual Cyp19-luc mice are highly responsive to aromatase inducers, suggesting this model, with further optimization, may have potential as an in vivo screening tool

Regulation of Bioluminescence in Photobacterium leiognathi Strain KNH6

PubMed Central

Rader, Bethany A.; Stabb, Eric V.; Mandel, Mark J.

2015-01-01

ABSTRACT Bacterial bioluminescence is taxonomically restricted to certain proteobacteria, many of which belong to the Vibrionaceae. In the most well-studied cases, pheromone signaling plays a key role in regulation of light production. However, previous reports have indicated that certain Photobacterium strains do not use this regulatory method for controlling luminescence. In this study, we combined genome sequencing with genetic approaches to characterize the regulation of luminescence in Photobacterium leiognathi strain KNH6, an extremely bright isolate. Using transposon mutagenesis and screening for decreased luminescence, we identified insertions in genes encoding components necessary for the luciferase reaction (lux, lum, and rib operons) as well as in nine other loci. These additional loci encode gene products predicted to be involved in the tricarboxylic acid (TCA) cycle, DNA and RNA metabolism, transcriptional regulation, and the synthesis of cytochrome c, peptidoglycan, and fatty acids. The mutagenesis screen did not identify any mutants with disruptions of predicted pheromone-related loci. Using targeted gene insertional disruptions, we demonstrate that under the growth conditions tested, luminescence levels do not appear to be controlled through canonical pheromone signaling systems in this strain. IMPORTANCE Despite the long-standing interest in luminous bacteria, outside a few model organisms, little is known about the regulation and function of luminescence. Light-producing marine bacteria are widely distributed and have diverse lifestyles, suggesting that the control and significance of luminescence may be similarly diverse. In this study, we apply genetic tools to the study of regulation of light production in the extremely bright isolate Photobacterium leiognathi KNH6. Our results suggest an unusual lack of canonical pheromone-mediated control of luminescence and contribute to a better understanding of alternative strategies for regulation of a

MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

PubMed Central

Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

2014-01-01

Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, S.B.; Halenda, S.P.; Bylund, D.B.

1991-02-01

The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipasemore » A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.« less

Turbulent entrainment across turbulent-nonturbulent interfaces in stably stratified mixing layers

NASA Astrophysics Data System (ADS)

Watanabe, T.; Riley, J. J.; Nagata, K.

2017-10-01

The entrainment process in stably stratified mixing layers is studied in relation to the turbulent-nonturbulent interface (TNTI) using direct numerical simulations. The statistics are calculated with the interface coordinate in an Eulerian frame as well as with the Lagrangian fluid particles entrained from the nonturbulent to the turbulent regions. The characteristics of entrainment change as the buoyancy Reynolds number Reb decreases and the flow begins to layer. The baroclinic torque delays the enstrophy growth of the entrained fluids at small Reb, while this effect is less efficient for large Reb. The entrained particle movement within the TNTI layer is dominated by the small dissipative scales, and the rapid decay of the kinetic energy dissipation rate due to buoyancy causes the entrained particle movement relative to the interface location to become slower. Although the Eulerian statistics confirm that there exists turbulent fluid with strong vorticity or with large buoyancy frequency near the TNTI, the entrained fluid particles circumvent these regions by passing through the TNTI in strain-dominant regions or in regions with small buoyancy frequency. The multiparticle statistics show that once the nonturbulent fluid volumes are entrained, they are deformed into flattened shapes in the vertical direction and diffuse in the horizontal direction. When Reb is large enough for small-scale turbulence to exist, the entrained fluid is able to penetrate into the turbulent core region. Once the flow begins to layer with decreasing Reb, however, the entrained fluid volume remains near the outer edge of the turbulent region and forms a stably stratified layer without vertical overturning.

[Activation of the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones in the presence of nitrofurans and NO generators].

PubMed

Zaĭtseva, Iu V; Granik, V G; Belik, A S; Koksharova, O A; Khmel', I A

2010-01-01

Nitrofurans (nitrofurazone, nitrofurantoin, furazidin, nifuroxazide), and nitric oxide generators (sodium nitroprusside and isosorbide mononitrate) in subinhibitory concentrations were shown to significantly increase the bioluminescence of the sensor Escherichia coli strains used for detecting N-acyl-homoserine lactones, signaling molecules of Quorum Sensing (QS) regulatory systems. The highest activation of bioluminescence (up to 250-400 fold) was observed in the presence of nitrofurazone on E. coli DH5alpha biosensors containing lux-reporter plasmids pSB401 or pSB536. However, this activation was not specifically associated with the functioning of QS systems. We suggest that the effect observed results from a direct action of nitrofurans and NO donors on the process of bioluminescence. The data indicate the necessity of using the biosensors that make it possible to detect specific effects of substances tested on QS regulation.

Sustained delivery of proangiogenic microRNA-132 by nanoparticle transfection improves endothelial cell transplantation

PubMed Central

Devalliere, Julie; Chang, William G.; Andrejecsk, Jillian W.; Abrahimi, Parwiz; Cheng, Christopher J.; Jane-wit, Dan; Saltzman, W. Mark; Pober, Jordan S.

2014-01-01

Transplantation of endothelial cells (ECs) for therapeutic vascularization or tissue engineering is a promising method for increasing tissue perfusion. Here, we report on a new approach for enhanced EC transplantation using targeted nanoparticle transfection to deliver proangiogenic microRNA-132 (miR-132) to cultured ECs before their transplantation, thereby sensitizing cells to the effects of endogenous growth factors. We synthesized biodegradable PLGA polymer nanoparticles (NPs) that were loaded with miR-132 and coated with cyclic RGD (cRGD) peptides that target integrin αvβ3 expressed on cultured human umbilical vein ECs (HUVECs), increasing NP uptake through clathrin-coated pits. Unlike previously reported NPs for miR delivery, these NPs slowly release RNA for several weeks. The endocytosed NPs remain in clathrin-coated vesicles from which they mediate intracellular delivery of siRNA or miRNA. Transfection of HUVECs with miR-132 enhances growth factor-induced proliferation and migration in 2D culture, producing a 1.8- and 5-fold increase, respectively. However, while the effects of conventional transfection were short-lived, NP transfection produced protein knockdown and biological effects that were significantly longer in duration (≥6 d). Transfection of HUVECs with miR-132 NP resulted in a 2-fold increase in the number of microvessels per square millimeter compared to lipid after transplantation into immunodeficient mice and led to a higher number of mural cell-invested vessels than control transfection. These data suggest that sustained delivery of miR-132 encapsulated in a targeted biodegradable polymer NP is a safe and efficient strategy to improve EC transplantation and vascularization.—Devalliere, J., Chang, W. G., Andrejecsk, J. W., Abrahimi, P., Cheng, C. J., Jane-wit, D., Saltzman, W. M., Pober, J. S. Sustained delivery of proangiogenic microRNA-132 by nanoparticle transfection improves endothelial cell transplantation. PMID:24221087

Tissue Engineering Using Transfected Growth-Factor Genes

NASA Technical Reports Server (NTRS)

Madry, Henning; Langer, Robert S.; Freed, Lisa E.; Trippel, Stephen; Vunjak-Novakovic, Gordana

2005-01-01

A method of growing bioengineered tissues includes, as a major component, the use of mammalian cells that have been transfected with genes for secretion of regulator and growth-factor substances. In a typical application, one either seeds the cells onto an artificial matrix made of a synthetic or natural biocompatible material, or else one cultures the cells until they secrete a desired amount of an extracellular matrix. If such a bioengineered tissue construct is to be used for surgical replacement of injured tissue, then the cells should preferably be the patient s own cells or, if not, at least cells matched to the patient s cells according to a human-leucocyteantigen (HLA) test. The bioengineered tissue construct is typically implanted in the patient's injured natural tissue, wherein the growth-factor genes enhance metabolic functions that promote the in vitro development of functional tissue constructs and their integration with native tissues. If the matrix is biodegradable, then one of the results of metabolism could be absorption of the matrix and replacement of the matrix with tissue formed at least partly by the transfected cells. The method was developed for articular chondrocytes but can (at least in principle) be extended to a variety of cell types and biocompatible matrix materials, including ones that have been exploited in prior tissue-engineering methods. Examples of cell types include chondrocytes, hepatocytes, islet cells, nerve cells, muscle cells, other organ cells, bone- and cartilage-forming cells, epithelial and endothelial cells, connective- tissue stem cells, mesodermal stem cells, and cells of the liver and the pancreas. Cells can be obtained from cell-line cultures, biopsies, and tissue banks. Genes, molecules, or nucleic acids that secrete factors that influence the growth of cells, the production of extracellular matrix material, and other cell functions can be inserted in cells by any of a variety of standard transfection techniques.

Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

PubMed Central

2010-01-01

Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival. PMID:20144189

Hybrid radiosity-SP3 equation based bioluminescence tomography reconstruction for turbid medium with low- and non-scattering regions

NASA Astrophysics Data System (ADS)

Chen, Xueli; Zhang, Qitan; Yang, Defu; Liang, Jimin

2014-01-01

To provide an ideal solution for a specific problem of gastric cancer detection in which low-scattering regions simultaneously existed with both the non- and high-scattering regions, a novel hybrid radiosity-SP3 equation based reconstruction algorithm for bioluminescence tomography was proposed in this paper. In the algorithm, the third-order simplified spherical harmonics approximation (SP3) was combined with the radiosity equation to describe the bioluminescent light propagation in tissues, which provided acceptable accuracy for the turbid medium with both low- and non-scattering regions. The performance of the algorithm was evaluated with digital mouse based simulations and a gastric cancer-bearing mouse based in situ experiment. Primary results demonstrated the feasibility and superiority of the proposed algorithm for the turbid medium with low- and non-scattering regions.

The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells.

PubMed

Jiwaji, Meesbah; Daly, Rónán; Pansare, Kshama; McLean, Pauline; Yang, Jingli; Kolch, Walter; Pitt, Andrew R

2010-12-31

The importance of appropriate normalization controls in quantitative real-time polymerase chain reaction (qPCR) experiments has become more apparent as the number of biological studies using this methodology has increased. In developing a system to study gene expression from transiently transfected plasmids, it became clear that normalization using chromosomally encoded genes is not ideal, at it does not take into account the transfection efficiency and the significantly lower expression levels of the plasmids. We have developed and validated a normalization method for qPCR using a co-transfected plasmid. The best chromosomal gene for normalization in the presence of the transcriptional activators used in this study, cadmium, dexamethasone, forskolin and phorbol-12-myristate 13-acetate was first identified. qPCR data was analyzed using geNorm, Normfinder and BestKeeper. Each software application was found to rank the normalization controls differently with no clear correlation. Including a co-transfected plasmid encoding the Renilla luciferase gene (Rluc) in this analysis showed that its calculated stability was not as good as the optimised chromosomal genes, most likely as a result of the lower expression levels and transfection variability. Finally, we validated these analyses by testing two chromosomal genes (B2M and ActB) and a co-transfected gene (Rluc) under biological conditions. When analyzing co-transfected plasmids, Rluc normalization gave the smallest errors compared to the chromosomal reference genes. Our data demonstrates that transfected Rluc is the most appropriate normalization reference gene for transient transfection qPCR analysis; it significantly reduces the standard deviation within biological experiments as it takes into account the transfection efficiencies and has easily controllable expression levels. This improves reproducibility, data validity and most importantly, enables accurate interpretation of qPCR data.

Selection of stably folded proteins by phage-display with proteolysis.

PubMed

Bai, Yawen; Feng, Hanqiao

2004-05-01

To facilitate the process of protein design and learn the basic rules that control the structure and stability of proteins, combinatorial methods have been developed to select or screen proteins with desired properties from libraries of mutants. One such method uses phage-display and proteolysis to select stably folded proteins. This method does not rely on specific properties of proteins for selection. Therefore, in principle it can be applied to any protein. Since its first demonstration in 1998, the method has been used to create hyperthermophilic proteins, to evolve novel folded domains from a library generated by combinatorial shuffling of polypeptide segments and to convert a partially unfolded structure to a fully folded protein.

Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells.

PubMed Central

Reeves, R; Gorman, C M; Howard, B

1985-01-01

The nucleoprotein structures formed on various plasmid expression vectors transfected into mammalian cells by both the calcium phosphate and DEAE-dextran methods have been studied. We demonstrate by a variety of means that mammalian cells are capable of rapidly assembling non-integrated circular plasmids (both replicating and non-replicating) into typical "minichromosomes" containing nucleosomes with a 190 bp repetitive spacing. Treatment of recipient cells with sodium butyrate for a short period of time (12-16 h) immediately following transfection markedly increased the DNase I digestion sensitivity of the newly assembled plasmid chromatin. Furthermore, minichromosomes isolated from such butyrate-treated cells are depleted in histone H1 and contain highly acetylated forms of histone H4. These findings are entirely consistent with our earlier speculation (Gorman et al., Nucleic Acids Res. 11, 1044; 1983) that appropriate butyrate treatment might stimulate transient expression of newly transfected genes by facilitating their assembly into an "active" type of chromatin structure. Images PMID:3859838

Localized transfection on arrays of magnetic beads coated with PCR products.

PubMed

Isalan, Mark; Santori, Maria Isabel; Gonzalez, Cayetano; Serrano, Luis

2005-02-01

High-throughput gene analysis would benefit from new approaches for delivering DNA or RNA into cells. Here we describe a simple system that allows any molecular biology laboratory to carry out multiple, parallel cell transfections on microscope coverslip arrays. By using magnetically defined positions and PCR product-coated paramagnetic beads, we achieved transfection in a variety of cell lines. Beads may be added to the cells at any time, allowing both spatial and temporal control of transfection. Because the beads may be coated with more than one gene construct, the method can be used to achieve cotransfection within single cells. Furthermore, PCR-generated mutants may be conveniently screened, bypassing cloning and plasmid purification steps. We illustrated the applicability of the method by screening combinatorial peptide libraries, fused to GFP, to identify previously unknown cellular localization motifs. In this way, we identified several localizing peptides, including structured localization signals based around the scaffold of a single C2H2 zinc finger.

Inner Ear Gene Transfection in Neonatal Mice Using Adeno-Associated Viral Vector: A Comparison of Two Approaches

PubMed Central

Xia, Li; Yin, Shankai; Wang, Jian

2012-01-01

Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant. PMID:22912830

Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone.

PubMed

Arts, Remco; den Hartog, Ilona; Zijlema, Stefan E; Thijssen, Vito; van der Beelen, Stan H E; Merkx, Maarten

2016-04-19

Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

Assessment of hospital daily cleaning practices using ATP bioluminescence in a developing country.

PubMed

Zambrano, Alejandra A; Jones, Alex; Otero, Paula; Ajenjo, Maria Cristina; Labarca, Jaime A

2014-01-01

Visual assessment of surfaces may not be enough to document the level of cleanliness in the hospital setting. It is necessary to introduce quantitative methods to document the results of this practice. To evaluate the efficacy of hospital terminal cleaning procedures, using an adenosine triphosphate (ATP) bioluminescence method in a teaching hospital. During 2008 we conducted an evaluation using ATP bioluminescence LIGHTNING MVP™ (Arquimed) of external and internal housekeeping service. After conducting an initial evaluation we implemented education of cleaning practices and finally we did a post intervention evaluation. Using chi-square method we compared prior versus after cleaning, quality of cleaning performed by external versus internal personnel, single versus double terminal cleaning procedures and prior versus after intervention. A finding of three RLU or less was considered a clean surface. We performed 198 evaluations in 33 patient units and nine OR. Internal personnel accomplished 25.37% of clean surfaces before and 80% after the education intervention (p=0.01). In contrast, external personnel obtained 68.8% before and 73.33% after intervention (p=0.3). This study suggests that visual assessment is not enough to ensure quality of the process and it is necessary to document the level of cleanliness by quantitative methods. Copyright © 2014 Elsevier Editora Ltda. All rights reserved.

Identification of a vacuolar proton channel that triggers the bioluminescent flash in dinoflagellates

PubMed Central

Rodriguez, Juan D.; Haq, Saddef; Bachvaroff, Tsvetan; Nowak, Kristine F.; Nowak, Scott J.; Morgan, Deri; Cherny, Vladimir V.; Sapp, Maredith M.; Bernstein, Steven; Bolt, Andrew; DeCoursey, Thomas E.; Place, Allen R.; Smith, Susan M. E.

2017-01-01

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel (HV1) that opens in response to depolarizing voltage across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thereby causing a pH drop that triggers light emission. HV1 channels were subsequently identified and demonstrated to have important functions in a multitude of eukaryotic cells. Here we report a predicted protein from Lingulodinium polyedrum that displays hallmark properties of bona fide HV1, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. Western blotting and fluorescence confocal microscopy of isolated L. polyedrum scintillons immunostained with antibody to LpHV1 confirm LpHV1’s predicted organellar location. Proteomics analysis demonstrates that isolated scintillon preparations contain peptides that map to LpHV1. Finally, Zn2+ inhibits both LpHV1 proton current and the acid-induced flash in isolated scintillons. These results implicate LpHV1 as the voltage gated proton channel that triggers bioluminescence in L. polyedrum, confirming Hastings’ hypothesis. The same channel likely mediates the action potential that communicates the signal along the tonoplast to the scintillon. PMID:28178296

Stabilizing in vitro ultrasound-mediated gene transfection by regulating cavitation.

PubMed

Lo, Chia-Wen; Desjouy, Cyril; Chen, Shing-Ru; Lee, Jyun-Lin; Inserra, Claude; Béra, Jean-Christophe; Chen, Wen-Shiang

2014-03-01

It is well known that acoustic cavitation can facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, partially due to the unstationary behavior of the initiation and leveling of cavitation, the sonoporation effect is usually unstable, especially in low intensity conditions. A system which is able to regulate the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop is implemented and its effect on in vitro gene transfection is tested. The regulated system provided better time stability and reproducibility of the cavitation levels than the unregulated conditions. Cultured hepatoma cells (BNL) mixed with 10 μg luciferase plasmids are exposed to 1-MHz pulsed ultrasound with or without cavitation regulation, and the gene transfection efficiency and cell viability are subsequently assessed. Experimental results show that for all exposure intensities (low, medium, and high), stable and intensity dependent, although not higher, gene expression could be achieved in the regulated cavitation system than the unregulated conditions. The cavitation regulation system provides a better control of cavitation and its bioeffect which are crucial important for clinical applications of ultrasound-mediated gene transfection. Copyright © 2013 Elsevier B.V. All rights reserved.

Biophysical and transfection studies of the diC(14)-amidine/DNA complex.

PubMed Central

Cherezov, Vadim; Qiu, Hong; Pector, Veronique; Vandenbranden, Michel; Ruysschaert, Jean-Marie; Caffrey, Martin

2002-01-01

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection. PMID:12023234

Application of nanostructured biochips for efficient cell transfection microarrays

NASA Astrophysics Data System (ADS)

Akkamsetty, Yamini; Hook, Andrew L.; Thissen, Helmut; Hayes, Jason P.; Voelcker, Nicolas H.

2007-01-01

Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart materials possessing both switchable and patterned surface properties. A system had been developed to spatially manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced. However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by contact printing has also been investigated.

Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).

PubMed

Gomaa, Fatma; Garcia, Paulo A; Delaney, Jennifer; Girguis, Peter R; Buie, Cullen R; Edgcomb, Virginia P

2017-09-01

We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: (1) a square-wave electroporator designed for eukaryotes, (2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and (3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (>10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transfected by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transfected with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Hyaluronidase and Collagenase Increase the Transfection Efficiency of Gene Electrotransfer in Various Murine Tumors

PubMed Central

Golzio, Muriel; Sersa, Gregor; Escoffre, Jean-Michel; Coer, Andrej; Vidic, Suzana; Teissie, Justin

2012-01-01

Abstract One of the applications of electroporation/electropulsation in biomedicine is gene electrotransfer, the wider use of which is hindered by low transfection efficiency in vivo compared with viral vectors. The aim of our study was to determine whether modulation of the extracellular matrix in solid tumors, using collagenase and hyaluronidase, could increase the transfection efficiency of gene electrotransfer in histologically different solid subcutaneous tumors in mice. Tumors were treated with enzymes before electrotransfer of plasmid DNA encoding either green fluorescent protein or luciferase. Transfection efficiency was determined 3, 9, and 15 days posttransfection. We demonstrated that pretreatment of tumors with a combination of enzymes significantly increased the transfection efficiency of electrotransfer in tumors with a high extracellular matrix area (LPB fibrosarcoma). In tumors with a smaller extracellular matrix area and less organized collagen lattice, the increase was not so pronounced (SA-1 fibrosarcoma and EAT carcinoma), whereas in B16 melanoma, in which only traces of collagen are present, pretreatment of tumors with hyaluronidase alone was more efficient than pretreatment with both enzymes. In conclusion, our results suggest that modification of the extracellular matrix could improve distribution of plasmid DNA in solid subcutaneous tumors, demonstrated by an increase in transfection efficiency, and thus have important clinical implications for electrogene therapy. PMID:21797718

DyNAvectors: dynamic constitutional vectors for adaptive DNA transfection.

PubMed

Clima, Lilia; Peptanariu, Dragos; Pinteala, Mariana; Salic, Adrian; Barboiu, Mihail

2015-12-25

Dynamic constitutional frameworks, based on squalene, PEG and PEI components, reversibly connected to core centers, allow the efficient identification of adaptive vectors for good DNA transfection efficiency and are well tolerated by mammalian cells.

Mechanisms of energy conversion and transfer in bioluminescence. Progress report, August 15, 1976--November 14, 1977. [Renilla (anthozoa)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cormier, M.J.

1977-01-01

Progress is reported on the following studies: isolation of luciferase and green fluorescent protein (GFP) from Renilla; chemical properties and chemical reactions of luciferase and GFP; and analogy of energy transfer in bioluminescence to energy transfer in photosynthesis. (HLW)

Use of inert gas jets to measure the forces required for mechanical gene transfection

PubMed Central

2012-01-01

Background Transferring genes and drugs into cells is central to how we now study, identify and treat diseases. Several non-viral gene therapy methods that rely on the mechanical disruption of the plasma membrane have been proposed, but the success of these methods has been limited due to a lack of understanding of the mechanical parameters that lead to cell membrane permeability. Methods We use a simple jet of inert gas to induce local transfection of plasmid DNA both in vitro (HeLa cells) and in vivo (chicken chorioallantoic membrane). Five different capillary tube inner diameters and three different gases were used to treat the cells to understand the dependency of transfection efficiency on the dynamic parameters. Results The simple setup has the advantage of allowing us to calculate the forces acting on cells during transfection. We found permeabilization efficiency was related to the dynamic pressure of the jet. The range of dynamic pressures that led to transfection in HeLa cells was small (200 ± 20 Pa) above which cell stripping occurred. We determined that the temporary pores allow the passage of dextran up to 40 kDa and reclose in less than 5 seconds after treatment. The optimized parameters were also successfully tested in vivo using the chorioallantoic membrane of the chick embryo. Conclusions The results show that the number of cells transfected with the plasmid scales with the dynamic pressure of the jet. Our results show that mechanical methods have a very small window in which cells are permeabilized without injury (200 to 290 Pa). This simple apparatus helps define the forces needed for physical cell transfection methods. PMID:22963645

[VEGF165 transfected endothelial progenitor cells mediated by lentivirus alleviated ALI in rats].

PubMed

He, Zhaohui; He, Huiwei; Lu, Yuanhua; Chen, Zhi; Xu, Fanghua; Wang, Rongsheng; Yang, Chunli

2017-11-01

To investigate the protective effects of vascular endothelial growth factor-165 (VEGF165) transfected the endothelial progenitor cells (EPCs) mediated by lentivirus on acute lung injury (ALI) in rats. The mononuclear cells from the male Sprague-Dawley (SD) rats were isolated and cultured to get the EPCs for study. The lentivirus vector carrying the human VEGF165 gene was constructed. According to the random number table method, 90 male SD rats were divided into ALI model group, phosphate buffer solution (PBS) group, EPCs treatment group, none transfected EPCs treatment group and VEGF165 transfected EPCs treatment group, and the rats in each group were subdivided into 4, 12 and 48 hours subgroups, with 6 rats in each subgroup. The rat model of ALI was reproduced by intravenous injection of oleic acid (0.15 μL/g). Then each treatment group was given PBS, EPCs, none transfected EPCs and VEGF165 transfected EPCs respectively with the same volume of 0.2 mL. For the groups with cells, about 1×10 6 cells were contained. Abdominal aortic blood and lung tissue were harvested at 4, 12 and 48 hours. Arterial blood gas analysis was performed. The lung wet/dry weight ratio (W/D) was calculated. The expressions of induced nitric oxide synthase (iNOS), endothelin-1 (ET-1) and VEGF165 were determined by enzyme-linked immunosorbent assay (ELISA). After dyed with hematoxylin-eosin (HE), the lung tissue pathology was observed and the lung injury score was performed. Compared with the ALI model group, the arterial partial pressure of oxygen (PaO 2 ) in EPCs, none transfected EPCs and VEGF165 transfected EPCs treatment groups was significantly increased from 4 hours, and lung W/D, expressions of iNOS and ET-1 were significantly decreased, and VEGF165 expression was significantly increased. Compared with the EPCs treatment group, the increase in PaO 2 , the decrease in lung W/D and expressions of iNOS and ET-1, and the increase in VEGF165 expression in VEGF165 transfected EPCs

Establishment and characterization of in vivo orthotopic bioluminescent xenograft models from human osteosarcoma cell lines in Swiss nude and NSG mice.

PubMed

Marques da Costa, Maria Eugenia; Daudigeos-Dubus, Estelle; Gomez-Brouchet, Anne; Bawa, Olivia; Rouffiac, Valerie; Serra, Massimo; Scotlandi, Katia; Santos, Conceição; Geoerger, Birgit; Gaspar, Nathalie

2018-03-01

Osteosarcoma is one of the most common primary bone tumors in childhood and adolescence. Metastases occurrence at diagnosis or during disease evolution is the main therapeutic challenge. New drug evaluation to improve patient survival requires the development of various preclinical models mimicking at best the complexity of the disease and its metastatic potential. We describe here the development and characteristics of two orthotopic bioluminescent (Luc/mKate2) cell-derived xenograft (CDX) models, Saos-2-B-Luc/mKate2-CDX and HOS-Luc/mKate2-CDX, in different immune (nude and NSG mouse strains) and bone (intratibial and paratibial with periosteum activation) contexts. IVIS SpectrumCT system allowed both longitudinal computed tomography (CT) and bioluminescence real-time follow-up of primary tumor growth and metastatic spread, which was confirmed by histology. The murine immune context influenced tumor engraftment, primary tumor growth, and metastatic spread to lungs, bone, and spleen (an unusual localization in humans). Engraftment in NSG mice was found superior to that found in nude mice and intratibial bone environment more favorable to engraftment compared to paratibial injection. The genetic background of the two CDX models also led to distinct primary tumor behavior observed on CT scan. Saos-2-B-Luc/mKate2-CDX showed osteocondensed, HOS-Luc/mKate2-CDX osteolytic morphology. Bioluminescence defined a faster growth of the primary tumor and metastases in Saos-2-B-Luc/mKate2-CDX than in HOS-Luc/mKate2-CDX. The early detection of primary tumor growth and metastatic spread by bioluminescence allows an improved exploration of osteosarcoma disease at tumor progression, and metastatic spread, as well as the evaluations of anticancer treatments. Our orthotopic models with metastatic spread bring complementary information to other types of existing osteosarcoma models. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency.

PubMed

Campos, Vinicius Farias; de Leon, Priscila Marques Moura; Komninou, Eliza Rossi; Dellagostin, Odir Antônio; Deschamps, João Carlos; Seixas, Fabiana Kömmling; Collares, Tiago

2011-11-01

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT. Copyright © 2011 Elsevier Inc. All rights reserved.

Inhibition of breast cancer metastasis by co-transfection of miR-31/193b-mimics

PubMed Central

Hashemi, Zahra Sadat; Moghadam, Mehdi Forouzandeh; Farokhimanesh, Samila; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

2018-01-01

Objective(s): Various studies have been conducted to reduce the metastatic behavior of cancerous cells. In this regard, ectopic expression of anti-metastatic microRNAs by miR-mimic and miR-restoration-based therapies could bring new insights to the field. In the present study, the consequences of co-transfecting breast cancer cell lines with miR-193b and miR-31 were investigated via invasion and migration assays. Materials and Methods: Double stranded oligonucleotide of mature miR-193b-3p and miR-31-5p were cloned into pcDNA 6.2gw/EmGFP plasmid. The resulting plasmids were used for transfection. Real time-PCR was performed to assess the expression of miR-193b and miR-31 as well as Ras homolog gene family member A (RhoA) and urokinase-type plasminogen activator (uPA) as miR targets. Scratch, Transwell migration and Matrigel invasion assays were carried out to assess the extent of migration and invasion of cell lines. Results: The most significant increase in expression of miRs belonged to the single transfection of mimic-miRs in MDA-MB231. Although the co-transfection was not as successful as single transfection in miR expression, it was significantly more effective in inhibition of the cells invasive potential. Conclusion: Although the miR-restoration therapy based on co-transfection of two miRs could be less effective in expression of each miRNA, the resulting decrease in metastatic behavior of the cells is more significant due to collective effect of co-transfection to decrease target gene expression. Our results revealed that employing this sort of combinatorial strategies could lead to more efficient reduction in metastatic behavior. It seems that using this strategy would bring about more successful therapeutic outcomes. PMID:29796229

The Use of Stimulable Bioluminescence From Dinoflagellates as a Means of Detecting Toxicity in the Marine Environment

DTIC Science & Technology

1993-03-01

tributyltin chloride (TFITCI), Copper (11) Sulfate (CuSO 4 I. zinc sulfate (ZnSO4 ), or storm drain effluent. Stimulable bioluminescence was measured at...to several metals and storm drain effluent. Dinoflagellate cells were exposed to various concentrations of tributyltin chloride (TBI1C), copper (II

Engineering the metal sensitive sites in Macrolampis sp2 firefly luciferase and use as a novel bioluminescent ratiometric biosensor for heavy metals.

PubMed

Gabriel, Gabriele V M; Viviani, Vadim R

2016-12-01

Most luminescent biosensors for heavy metals are fluorescent and rely on intensity measurements, whereas a few are ratiometric and rely on spectral changes. Bioluminescent biosensors for heavy metals are less common. Firefly luciferases have been coupled to responsive promoters for mercury and arsenium, and used as light on biosensors. Firefly luciferase bioluminescence spectrum is naturally sensitive to heavy metal cations such as zinc and mercury and to pH. Although pH sensitivity of firefly luciferases was shown to be useful for ratiometric estimation of intracellular pH, its potential use for ratiometric estimation of heavy metals was never considered. Using the yellow-emitting Macrolampis sp2 firefly luciferase and site-directed mutagenesis, we show that the residues H310 and E354 constitute two critical sites for metal sensitivity that can be engineered to increase sensitivity to zinc, nickel, and mercury. A linear relationship between cation concentration and the ratio of bioluminescence intensities at 550 and 610 nm allowed, for the first time, the ratiometric estimation of heavy metals concentrations down to 0.10 mM, demonstrating the potential applicability of firefly luciferases as enzymatic and intracellular ratiometric metal biosensors.

Enhanced photo-transfection efficiency of mammalian cells on graphene coated substrates

NASA Astrophysics Data System (ADS)

Mthunzi, Patience; He, Kuang; Ngcobo, Sandile; Warner, Jamie W.

2014-03-01

Literature reports graphene, an atomic-thick sheet of carbon atoms as one of the promising biocompatible scaffolds that promotes cellular proliferation in human mesenchymal stem cells. On the other hand, different mammalian cell lines including the induced pluripotent stem cells exhibited an accelerated proliferation rate when cultured on graphene or graphene oxide coated substrates. These findings provide strong motivation to explore the full capability of graphene in further pluripotent stem cell research activities as there exists an urgent requirement to preserve their therapeutic potential. This therefore calls for non-invasive procedures for handling stem cells in-vitro. For example, resent literature has shown successful laser light driven transfection in both multipotent and pluripotent stem cells. In order to explore the non-invasive nature of optical transfection alongside biocompatible qualities of graphene, in this work we investigated the impact of optically transfecting mouse embryonic stem (mES) cells plated on graphene coated sample chambers. Using Chinese Hamster Ovary cells (CHO-K1), we further studied the influence of graphene on cell viability as well as cell cytotoxicity through assessing changes in levels of mitochondrial adenosine triphosphate (ATP) activity and the release of cytosolic lactate dehydrogenase (LHD) respectively. Our results showed that compared to those treated on plain glass, CHO-K1 cells optically treated while plated on graphene coated substrates exhibited a higher production of ATP and a milder release of LDH. In addition there was enhanced photo-transfection efficiency in both CHO-K1 and mES cells irradiated on graphene sample chambers.

Mechanism of attenuation of a chimeric influenza A/B transfectant virus.

PubMed

Luo, G; Bergmann, M; Garcia-Sastre, A; Palese, P

1992-08-01

The ribonucleoprotein transfection system for influenza virus allowed us to construct an influenza A virus containing a chimeric neuraminidase (NA) gene in which the noncoding sequence is derived from the NS gene of influenza B virus (T. Muster, E. K. Subbarao, M. Enami, B. P. Murphy, and P. Palese, Proc. Natl. Acad. Sci. USA 88:5177-5181, 1991). This transfectant virus is attenuated in mice and grows to lower titers in tissue culture than wild-type virus. Since such a virus has characteristics desirable for a live attenuated vaccine strain, attempts were made to characterize this virus at the molecular level. Our analysis suggests that the attenuation of the virus is due to changes in the cis signal sequences, which resulted in a reduction of transcription and replication of the chimeric NA gene. The major finding concerns a sixfold reduction in NA-specific viral RNA in the virion, causing a reduction in the ratio of infectious particles to physical particles compared with the ratio in wild-type virus. Although the NA-specific mRNA level is also reduced in transfectant virus-infected cells, it does not appear to contribute to the attenuation characteristics of the virus. The levels of the other RNAs and their expression appear to be unchanged for the transfectant virus. It is suggested that downregulation of the synthesis of one viral RNA segment leads to the generation of defective viruses during each replication cycle. We believe that this represents a general principle for attenuation which may be applied to other segmented viruses containing either single-stranded or double-stranded RNA.

Mechanism of gene transfection by polyamidoamine (PAMAM) dendrimers modified with ornithine residues.

PubMed

Kumar, Ajay; Yellepeddi, Venkata K; Vangara, Kiran K; Strychar, Kevin B; Palakurthi, Srinath

2011-11-01

The aim of this study was to prepare and investigate the mechanism of uptake of the dendriplexes prepared with ornithine-conjugated polyamidoamine (PAMAM) G4 dendrimers. Ornithine-conjugated PAMAMG4 dendrimers were prepared by Fmoc synthesis. A comparative transfection study in NCI H157G cells and polyamine transport-deficient cell line NCI H157R was performed to confirm the role of the polyamine transporter system (PAT) in the dendriplex uptake. Transfection efficiency significantly increased with increase in generation number and extent of ornithine conjugation. Transfection efficiency of the PAMAMG4-ORN60 dendrimers significantly decreased in presence of excess of ornithine (P < 0.05) and paraquat (P < 0.01) but not of PAMAMG4 dendrimers. Transfection efficiency of PAMAMG4-ORN60 was significantly low in NCI H157R (31.66 ± 3.95%, RFU: 17.87 ± 1.34) as compared to NCI H157G cell line (63.07 ± 6.8%, relative fluorescence units (RFU): 23.28 ± 0.66). Results indicate the role of PAT in addition to charge-mediated endocytosis in the internalization of ornithine-conjugated PAMAMG4 dendrimers. Cytotoxicity analysis (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay) in human embryonic kidney cell line (HEK) 293T cells showed that the dendriplexes were non-toxic at N/P 10.

Rigid aromatic linking moiety in cationic lipids for enhanced gene transfection efficiency.

PubMed

Wang, Bing; Zhao, Rui-Mo; Zhang, Ji; Liu, Yan-Hong; Huang, Zheng; Yu, Qing-Ying; Yu, Xiao-Qi

2017-08-18

Although numerous cationic lipids have been developed as non-viral gene vectors, the structure-activity relationship (SAR) of these materials remains unclear and needs further investigation. In this work, a series of lysine-derived cationic lipids containing linkages with different rigidity were designed and synthesized. SAR studies showed that lipids with rigid aromatic linkage could promote the formation of tight liposomes and enhance DNA condensation, which is essential for the gene delivery process. These lipids could give much higher transfection efficiency than those containing more flexible aliphatic linkage in various cell lines. Moreover, the rigid aromatic linkage also affords the material higher serum tolerance ability. Flow cytometry assay revealed that the target lipids have good cellular uptake, while confocal microscopy observation showed weaker endosome escape than Lipofectamine 2000. To solve such problem and further increase the transfection efficiency, some lysosomotropic reagents were used to improve the endosome escape of lipoplex. As expected, higher transfection efficiency than Lipofectamine 2000 could be obtained via this strategy. Cytotoxicity assay showed that these lipids have lower toxicity in various cell lines than Lipofectamine 2000, suggesting their potential for further application. This work demonstrates that a rigid aromatic linkage might distinctly improve the gene transfection abilities of cationic lipids and affords information to construct safe and efficient gene vector towards practical application. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Computational investigation of the effect of pH on the color of firefly bioluminescence by DFT.

PubMed

Pinto da Silva, Luís; Esteves da Silva, Joaquim C G

2011-04-04

In spite of recent advances towards understanding the mechanism of firefly bioluminescence, there is no consensus about which oxyluciferin (OxyLH(2)) species are the red and yellow-green emitters. The crystal structure of Luciola cruciata luciferase (LcLuc) revealed different conformations for the various steps of the bioluminescence reaction, with different degrees of polarity and rigidity of the active-site microenvironment. In this study, these different conformations of luciferase (Luc) are simulated and their effects on the different chemical equilibria of OxyLH(2) are investigated as a function of pH by means of density functional theory with the PBE0 functional. In particular, the thermodynamic properties and the absorption spectra of each species, as well as their relative stabilities in the ground and excited states, were computed in the different conformations of Luc. From the calculations it is possible to derive the acid dissociation and tautomeric constants, and the corresponding distribution diagrams. It is observed that the anionic keto form of OxyLH(2) is both the red and the yellow-green emitter. Consequently, the effect of Luc conformations on the structural and electronic properties of the Keto-(-1) form are studied. Finally, insights into the Luc-catalyzed light-emitting reaction are derived from the calculations. The multicolor bioluminescence can be explained by interactions of the emitter with active-site molecules, the effects of which on light emission are modulated by the internal dielectric constant of the different conformations. These interactions can suffer also from rearrangement due to entry of external solvent and changes in the protonation state of some amino acid residues and adenosine monophosphate (AMP). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Red Fluorescent Protein-Aequorin Fusions as Improved Bioluminescent Ca2+ Reporters in Single Cells and Mice

PubMed Central

Bakayan, Adil; Vaquero, Cecilia F.; Picazo, Fernando; Llopis, Juan

2011-01-01

Bioluminescence recording of Ca2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca2+ in various cell compartments. In addition, they would also serve to monitor Ca2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tdTA. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca2+ sensor reported to date and is, therefore, a promising probe to study Ca2+ dynamics in whole organisms or tissues expressing the transgene. PMID:21589654

Large-Scale Transient Transfection of Chinese Hamster Ovary Cells in Suspension.

PubMed

Rajendra, Yashas; Balasubramanian, Sowmya; Hacker, David L

2017-01-01

We describe a one-liter transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells using polyethyleneimine (PEI) for DNA delivery. The method involves transfection at a high cell density (5 × 10 6 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. We also describe an alternative method in which 90% of the pDNA is replaced by nonspecific (filler) DNA, and the production phase is performed at 31 °C in the presence of 0.25% N, N-dimethylacetamide (DMA).

Synthesis and aggregation properties of dissymmetric phytanyl-gemini surfactants for use as improved DNA transfection vectors.

PubMed

Wang, Haitang; Wettig, Shawn D

2011-01-14

Improvements in transfection efficiency are required in order to make the goal of cellular gene delivery by non-viral vectors realizable. Novel derivatives of gemini surfactants having dissymmetric tail groups have been designed specifically as a means to improve DNA transfection; the micelle and interfacial properties are reported herein. The effect of these substitutions on the aggregation properties of the gemini surfactants is discussed in the context of results for the m-3-m gemini series, previously reported in the literature. Phytanyl substitution results in lower cmc and higher micelle ionization. In addition, the phytanyl substituted gemini surfactants form vesicles at room temperature. Preliminary in vitro transfection assays showed the phytanyl substituted gemini surfactants to be more efficient transfection vectors as compared to symmetric gemini surfactants.

Modelling chemical reactions by QM/MM calculations: the case of the tautomerization in fireflies bioluminescent systems

NASA Astrophysics Data System (ADS)

Berraud-Pache, Romain; Garcia-Iriepa, Cristina; Navizet, Isabelle

2018-04-01

In less than half a century, the hybrid QM/MM method has become one of the most used technique to model molecules embedded in a complex environment. A well-known application of the QM/MM method is for biological systems. Nowadays, one can understand how enzymatic reactions work or compute spectroscopic properties, like the wavelength of emission. Here, we have tackled the issue of modelling chemical reactions inside proteins. We have studied a bioluminescent system, fireflies, and deciphered if a keto-enol tautomerization is possible inside the protein. The two tautomers are candidates to be the emissive molecule of the bioluminescence but no outcome has been reached. One hypothesis is to consider a possible keto-enol tautomerization to treat this issue, as it has been already observed in water. A joint approach combining extensive MD simulations as well as computation of key intermediates like TS using QM/MM calculations is presented in this publication. We also emphasize the procedure and difficulties met during this approach in order to give a guide for this kind of chemical reactions using QM/MM methods.

Modeling Chemical Reactions by QM/MM Calculations: The Case of the Tautomerization in Fireflies Bioluminescent Systems

PubMed Central

Berraud-Pache, Romain; Garcia-Iriepa, Cristina; Navizet, Isabelle

2018-01-01

In less than half a century, the hybrid QM/MM method has become one of the most used technique to model molecules embedded in a complex environment. A well-known application of the QM/MM method is for biological systems. Nowadays, one can understand how enzymatic reactions work or compute spectroscopic properties, like the wavelength of emission. Here, we have tackled the issue of modeling chemical reactions inside proteins. We have studied a bioluminescent system, fireflies, and deciphered if a keto-enol tautomerization is possible inside the protein. The two tautomers are candidates to be the emissive molecule of the bioluminescence but no outcome has been reached. One hypothesis is to consider a possible keto-enol tautomerization to treat this issue, as it has been already observed in water. A joint approach combining extensive MD simulations as well as computation of key intermediates like TS using QM/MM calculations is presented in this publication. We also emphasize the procedure and difficulties met during this approach in order to give a guide for this kind of chemical reactions using QM/MM methods. PMID:29719820

Modeling Chemical Reactions by QM/MM Calculations: The Case of the Tautomerization in Fireflies Bioluminescent Systems.

PubMed

Berraud-Pache, Romain; Garcia-Iriepa, Cristina; Navizet, Isabelle

2018-01-01

In less than half a century, the hybrid QM/MM method has become one of the most used technique to model molecules embedded in a complex environment. A well-known application of the QM/MM method is for biological systems. Nowadays, one can understand how enzymatic reactions work or compute spectroscopic properties, like the wavelength of emission. Here, we have tackled the issue of modeling chemical reactions inside proteins. We have studied a bioluminescent system, fireflies, and deciphered if a keto-enol tautomerization is possible inside the protein. The two tautomers are candidates to be the emissive molecule of the bioluminescence but no outcome has been reached. One hypothesis is to consider a possible keto-enol tautomerization to treat this issue, as it has been already observed in water. A joint approach combining extensive MD simulations as well as computation of key intermediates like TS using QM/MM calculations is presented in this publication. We also emphasize the procedure and difficulties met during this approach in order to give a guide for this kind of chemical reactions using QM/MM methods.

Bacteria meets influenza A virus: A bioluminescence mouse model of Escherichia coli O157:H7 following influenza A virus/Puerto Rico/8/34 (H1N1) strain infection.

PubMed

Wang, Zhongyi; Chi, Hang; Wang, Xiwen; Li, Wenliang; Li, Zhiping; Li, Jiaming; Fu, Yingying; Lu, Bing; Xia, Zhiping; Qian, Jun; Liu, Linna

2018-01-01

Objective To develop a bioluminescence-labelled bacterial infection model to monitor the colonization and clearance process of Escherichia coli O157:H7 in the lungs of mice following influenza A virus/Puerto Rico/8/34 (H1N1) strain (IAV/PR8) infection. Methods BALB/c mice were administered IAV/PR8 or 0.01 M phosphate-buffered saline (PBS; pH 7.4) intranasally 4 days prior to intranasal administration of 1 × 10 7 colony-forming units (CFU) of E. coli O157:H7-lux. Whole-body bioluminescent signals were monitored at 10 min, 4 h, 8 h, 12 h, 16 h and 24 h post-bacterial infection. Lung bioluminescent signals and bacterial load (CFU/g) were monitored at 4 h, 8 h, 12 h, 16 h and 24 h post-bacterial infection. Results Prior IAV/PR8 infection of mice resulted in a higher level of bacterial colonization and a lower rate of bacterial clearance from the lungs compared with mice treated with PBS. There were also consistent findings between the bioluminescence imaging and the CFU measurements in terms of identifying bacterial colonization and monitoring the clearance dynamics of E. coli O157:H7-lux in mouse lungs. Conclusion This novel bioluminescence-labelled bacterial infection model rapidly detected bacterial colonization of the lungs and monitored the clearance dynamics of E. coli O157:H7-lux following IAV/PR8 infection.

Integrated CMOS photodetectors and signal processing for very low-level chemical sensing with the bioluminescent bioreporter integrated circuit

NASA Technical Reports Server (NTRS)

Bolton, Eric K.; Sayler, Gary S.; Nivens, David E.; Rochelle, James M.; Ripp, Steven; Simpson, Michael L.

2002-01-01

We report an integrated CMOS microluminometer optimized for the detection of low-level bioluminescence as part of the bioluminescent bioreporter integrated circuit (BBIC). This microluminometer improves on previous devices through careful management of the sub-femtoampere currents, both signal and leakage, that flow in the front-end processing circuitry. In particular, the photodiode is operated with a reverse bias of only a few mV, requiring special attention to the reset circuitry of the current-to-frequency converter (CFC) that forms the front-end circuit. We report a sub-femtoampere leakage current and a minimum detectable signal (MDS) of 0.15 fA (1510 s integration time) using a room temperature 1.47 mm2 CMOS photodiode. This microluminometer can detect luminescence from as few as 5000 fully induced Pseudomonas fluorescens 5RL bacterial cells. c2002 Elsevier Science B.V. All rights reserved.

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses (host range restriction/Fv-1 gene/DNA transfection)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, I.C.; Yang, W.K.; Tennant, R.W.

1978-03-01

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-1/sup n/ (NIH-3T3) and two Fv-1/sup b/ (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an (N-tropic MuLV)SC-1 cell DNA preparation was slightly more infectious than a (B-tropic MuLV)SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 geonotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced bymore » the infected SC-I cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the (B-tropic MuLV)SC-1 cell DNA and the (N-tropic MuLV)SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.« less

Effects of photodynamic therapy on Gram-positive and Gram-negative bacterial biofilms by bioluminescence imaging and scanning electron microscopic analysis.

PubMed

Garcez, Aguinaldo S; Núñez, Silvia C; Azambuja, Nilton; Fregnani, Eduardo R; Rodriguez, Helena M H; Hamblin, Michael R; Suzuki, Hideo; Ribeiro, Martha S

2013-11-01

The aim of this study was to test photodynamic therapy (PDT) as an alternative approach to biofilm disruption on dental hard tissue, We evaluated the effect of methylene blue and a 660 nm diode laser on the viability and architecture of Gram-positive and Gram-negative bacterial biofilms. Ten human teeth were inoculated with bioluminescent Pseudomonas aeruginosa or Enterococcus faecalis to form 3 day biofilms in prepared root canals. Bioluminescence imaging was used to serially quantify and evaluate the bacterial viability, and scanning electron microscopic (SEM) imaging was used to assess architecture and morphology of bacterial biofilm before and after PDT employing methylene blue and 40 mW, 660 nm diode laser light delivered into the root canal via a 300 μm fiber for 240 sec, resulting in a total energy of 9.6 J. The data were statistically analyzed with analysis of variance (ANOVA) followed by Tukey test. The bacterial reduction showed a dose dependence; as the light energy increased, the bioluminescence decreased in both planktonic suspension and in biofilms. The SEM analysis showed a significant reduction of biofilm on the surface. PDT promoted disruption of the biofilm and the number of adherent bacteria was reduced. The photodynamic effect seems to disrupt the biofilm by acting both on bacterial cells and on the extracellular matrix.

Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

PubMed Central

Throm, Robert E.; Ouma, Annastasia A.; Zhou, Sheng; Chandrasekaran, Anantharaman; Lockey, Timothy; Greene, Michael; De Ravin, Suk See; Moayeri, Morvarid; Malech, Harry L.; Sorrentino, Brian P.

2009-01-01

Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common γ chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 × 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1). PMID:19286997

A novel mouse model of soft-tissue infection using bioluminescence imaging allows noninvasive, real-time monitoring of bacterial growth.

PubMed

Yoshioka, Kenji; Ishii, Ken; Kuramoto, Tetsuya; Nagai, Shigenori; Funao, Haruki; Ishihama, Hiroko; Shiono, Yuta; Sasaki, Aya; Aizawa, Mamoru; Okada, Yasunori; Koyasu, Shigeo; Toyama, Yoshiaki; Matsumoto, Morio

2014-01-01

Musculoskeletal infections, including surgical-site and implant-associated infections, often cause progressive inflammation and destroy areas of the soft tissue. Treating infections, especially those caused by multi-antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) remains a challenge. Although there are a few animal models that enable the quantitative evaluation of infection in soft tissues, these models are not always reproducible or sustainable. Here, we successfully established a real-time, in vivo, quantitative mouse model of soft-tissue infection in the superficial gluteus muscle (SGM) using bioluminescence imaging. A bioluminescent strain of MRSA was inoculated into the SGM of BALB/c adult male mice, followed by sequential measurement of bacterial photon intensity and serological and histological analyses of the mice. The mean photon intensity in the mice peaked immediately after inoculation and remained stable until day 28. The serum levels of interleukin-6, interleukin-1 and C-reactive protein at 12 hours after inoculation were significantly higher than those prior to inoculation, and the C-reactive protein remained significantly elevated until day 21. Histological analyses showed marked neutrophil infiltration and abscesses containing necrotic and fibrous tissues in the SGM. With this SGM mouse model, we successfully visualized and quantified stable bacterial growth over an extended period of time with bioluminescence imaging, which allowed us to monitor the process of infection without euthanizing the experimental animals. This model is applicable to in vivo evaluations of the long-term efficacy of novel antibiotics or antibacterial implants.

A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

PubMed

Rodgers, K K; Villey, I J; Ptaszek, L; Corbett, E; Schatz, D G; Coleman, J E

1999-07-15

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

Turbulent circulation above the surface heat source in a stably stratified environment

NASA Astrophysics Data System (ADS)

Kurbatskii, A. F.; Kurbatskaya, L. I.

2016-09-01

The results of the numerical modeling of turbulent structure of the penetrating convection above the urban heat island with a small aspect ratio in a stably stratified medium at rest are presented. The gradient diffusion representations for turbulent momentum and heat fluxes are used, which depend on three parameters — the turbulence kinetic energy, the velocity of its spectral expenditure, and the dispersion of temperature fluctuations. These parameters are found from the closed differential equations of balance in the RANS approach of turbulence description. The distributions of averaged velocity and temperature fields as well as turbulent characteristics agree well with measurement data.

Poly(β-Amino Ester)-Nanoparticle Mediated Transfection of Retinal Pigment Epithelial Cells In Vitro and In Vivo

PubMed Central

Bhutto, Imran; Handa, James T.; Green, Jordan J.

2012-01-01

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer∶DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×103-fold and 1.5±0.7×103-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p = 0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye

Novel dextran-spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers.

PubMed

Eliyahu, H; Makovitzki, A; Azzam, T; Zlotkin, A; Joseph, A; Gazit, D; Barenholz, Y; Domb, A J

2005-03-01

Recently, a novel cationic polymer, dextran-spermine (D-SPM) was developed for gene delivery. An efficient transfection was obtained using this polycation for a variety of genes and cell lines in serum-free or serum-poor medium. However, transfection using the water-soluble D-SPM-based polyplexes decreased with increasing serum concentration in cell culture in a concentration-dependent manner, reaching 95% inhibition at 50% serum in the cell growth medium. In order to overcome this obstacle, oleyl derivatives of D-SPM (which form micelles in aqueous phase) were synthesized at 1, 10, and 20 mol% of oleyl moiety to polymer epsilon-NH2 to form N-oleyl-D-SPM (ODS). Polyplexes based on ODS transfected well in medium containing 50% serum. Comparison with polyplexes based on well-established polymers (branched and linear polyethyleneimine) and with DOTAP/Cholesterol lipoplexes showed that regarding beta-galactosidase transgene expression level and cytotoxicity in tissue culture, the D-SPM and ODS compare well with the above polyplexes and lipoplexes. Intracellular trafficking using FITC-labeled ODS and Rhodamine-labeled pGeneGrip plasmid cloned with hBMP2 monitored by confocal microscopy revealed that during the transfection process the fluorescent-labeled polymer concentrates in the Golgi apparatus and around the nucleus, while the cell cytoplasm was free of fluorescent particles, suggesting that the polyplexes move in the cell toward the nucleus by vesicular transport through the cytoplasm and not by a random diffusion. We found that the plasmids penetrate the cell nucleus without the polymer. Preliminary results in zebra fish and mice demonstrate the potential of ODS to serve as an efficient nonviral vector for in vivo transfection.

Single-cell optoporation and transfection using femtosecond laser and optical tweezers.

PubMed

Waleed, Muhammad; Hwang, Sun-Uk; Kim, Jung-Dae; Shabbir, Irfan; Shin, Sang-Mo; Lee, Yong-Gu

2013-01-01

In this paper, we demonstrate a new single-cell optoporation and transfection technique using a femtosecond Gaussian laser beam and optical tweezers. Tightly focused near-infrared (NIR) femtosecond laser pulse was employed to transiently perforate the cellular membrane at a single point in MCF-7 cancer cells. A distinct technique was developed by trapping the microparticle using optical tweezers to focus the femtosecond laser precisely on the cell membrane to puncture it. Subsequently, an external gene was introduced in the cell by trapping and inserting the same plasmid-coated microparticle into the optoporated cell using optical tweezers. Various experimental parameters such as femtosecond laser exposure power, exposure time, puncture hole size, exact focusing of the femtosecond laser on the cell membrane, and cell healing time were closely analyzed to create the optimal conditions for cell viability. Following the insertion of plasmid-coated microparticles in the cell, the targeted cells exhibited green fluorescent protein (GFP) under the fluorescent microscope, hence confirming successful transfection into the cell. This new optoporation and transfection technique maximizes the level of selectivity and control over the targeted cell, and this may be a breakthrough method through which to induce controllable genetic changes in the cell.

Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

PubMed

Viviani, V R; Simões, A; Bevilaqua, V R; Gabriel, G V M; Arnoldi, F G C; Hirano, T

2016-08-30

Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also

Stably maintained dendritic spines are associated with lifelong memories

PubMed Central

Yang, Guang; Pan, Feng; Gan, Wen-Biao

2016-01-01

Changes in synaptic connections are considered essential for learning and memory formation1–6. However, it is unknown how neural circuits undergo continuous synaptic changes during learning while maintaining lifelong memories. Here we show, by following postsynaptic dendritic spines over time in the mouse cortex7–8, that learning and novel sensory experience lead to spine formation and elimination by a protracted process. The extent of spine remodelling correlates with behavioural improvement after learning, suggesting a crucial role of synaptic structural plasticity in memory formation and storage. Importantly, a small fraction of new spines induced by novel experience, together with most spines formed early during development and surviving experience-dependent elimination, are preserved throughout the entire life of an animal. These studies indicate that learning and daily sensory experience leave minute but permanent marks on cortical connections and suggest that lifelong memories are stored in largely stably connected synaptic networks. PMID:19946265

Microstructure of Turbulence in the Stably Stratified Boundary Layer

NASA Astrophysics Data System (ADS)

Sorbjan, Zbigniew; Balsley, Ben B.

2008-11-01

The microstructure of a stably stratified boundary layer, with a significant low-level nocturnal jet, is investigated based on observations from the CASES-99 campaign in Kansas, U.S.A. The reported, high-resolution vertical profiles of the temperature, wind speed, wind direction, pressure, and the turbulent dissipation rate, were collected under nocturnal conditions on October 14, 1999, using the CIRES Tethered Lifting System. Two methods for evaluating instantaneous (1-sec) background profiles are applied to the raw data. The background potential temperature is calculated using the “bubble sort” algorithm to produce a monotonically increasing potential temperature with increasing height. Other scalar quantities are smoothed using a running vertical average. The behaviour of background flow, buoyant overturns, turbulent fluctuations, and their respective histograms are presented. Ratios of the considered length scales and the Ozmidov scale are nearly constant with height, a fact that can be applied in practice for estimating instantaneous profiles of the dissipation rate.

In vitro energy transfer in Renilla bioluminescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, W.W.; Cormier, M.J.

1976-09-23

A quantitative study of in vitro energy transfer in a natural biological system is reported. The in vitro bioluminescent oxidation of Renilla (sea pansy) luciferin by luciferase produces a broad, structureless emission, peaking in the blue at 490 nm. In contrast, the live animal produces a structured emission peaking in the green at 509 nm. This difference in emission characteristics is due to the presence, in Renilla, of a green fluorescent protein (GFP). Addition of GFP in vitro sensitizes the oxyluciferin product excited state, resulting in the narrow, structured green emission characteristic of GFP fluorescence (lambda/sub max/ 509 nm). Undermore » conditions of efficient in vitro energy transfer (2.7 x 10/sup -6/ M GFP) the radiative quantum yield (with respect to luciferin) increases 5.7-fold from 5.3% (blue pathway) to 30% (green pathway). The fluorescence quantum yield of the Renilla GFP has been measured as 30%; thus, within the precision of our measurements (15% coefficient of variation) the in vitro energy transfer efficiency is a surprising 100%.« less

Hormonal induction of transfected genes depends on DNA topology.

PubMed Central

Piña, B; Haché, R J; Arnemann, J; Chalepakis, G; Slater, E P; Beato, M

1990-01-01

Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors. Images PMID:2153920

Pressure-Mediated Oligonucleotide Transfection of Rat and Human Cardiovascular Tissues

NASA Astrophysics Data System (ADS)

Mann, Michael J.; Gibbons, Gary H.; Hutchinson, Howard; Poston, Robert S.; Hoyt, E. Grant; Robbins, Robert C.; Dzau, Victor J.

1999-05-01

The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibited target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipulation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

Nonviral transfection of suspension cells in ultrasound standing wave fields.

PubMed

Lee, Yu-Hsiang; Peng, Ching-An

2007-05-01

Ultrasound-induced cavitation has been widely used for delivering DNA vectors into cells. However, this approach may seriously disrupt cell membranes and cause lethal damage when cells are exposed to the inertial cavitation field. In this study, instead of using sonoporation, ultrasound standing wave fields (USWF) were explored for nonviral transfection of suspension cells. Acoustic resonance in a tubular chamber was generated from the interference of waves emitted from a piezoelectric transducer and consequently reflected from a borosilicate glass coverslip. The suspended K562 erythroleukemia cells were transfected by polyethyleneimine (PEI)/DNA complexes with and without exposure to 1-MHz USWF for 5 min. During USWF exposure, K562 cells moved to the pressure nodal planes first and formed cell bands by the primary radiation force. Nanometer-sized PEI/DNA complexes, circulated between nodal planes by acoustic microstreaming, then used the cell agglomerates as the nucleating sites on which to attach. After incubation at 37 degrees C for 48 h, the efficiency of nonviral transfection based on EGFP transgene expression was determined by fluorescent microscopy and fluorometry. Both studies showed that USWF brought suspended K562 cells and PEI/DNA complexes into close contact at the pressure nodal planes, yielding an approximately 10-fold increment of EGFP transgene expression compared with the group without ultrasonic treatment.

Assessment of new biocompatible poly(N-(morpholino)ethyl methacrylate)-based copolymers by transfection of immortalized keratinocytes.

PubMed

Van Overstraeten-Schlögel, Nancy; Shim, Yong-Ho; Tevel, Virginie; Piel, Géraldine; Piette, Jacques; Dubois, Philippe; Raes, Martine

2012-02-01

Skin carcinomas are among the most commonly diagnosed tumors in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using the antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based copolymers. In order to improve the transfection efficiency of the classic PDMAEMA polymers, copolymers were synthesized including a poly(N-morpholino)ethylmethacrylate) (PMEMA) moiety for an improved proton-sponge effect, intended to favour the release of the oligonucleotide from the acidic endosome. These copolymers were synthesized either statistically (with alternating PDMAEMA and PMEMA fragments) or in blocks (one PDMAEMA block followed by one PMEMA block). MTT assays were performed using the PDMAEMA-PMEMA copolymers and revealed no significant cytotoxicity of these polymers at an N/P ratio of 7.3. Using fluorescent oligonucleotides and analyzing transfection efficiency by flow cytometry, we noticed no significant differences between the two kinds of copolymers. However copolymers with a higher DMAEMA content and a higher Mn were also those displaying the highest vectorization efficiency. Confocal microscopy showed that these copolymers induced a fine granular distribution of the transfected antisense oligonucleotides inside the cells. We also assessed the functionality of the transfected antisense oligonucleotide by transfecting immortalized GFP expressing keratinocytes with a GFP antisense oligonucleotide using these copolymers. A significant silencing was achieved with a PDMAEMA-PMEMA in block copolymer (Mn=41,000, 89 % PDMAEMA). Together, these results suggest that PDMAEMA-PMEMA copolymers combining low toxicity, vectorization and proton sponge properties, can be efficiently used to transfect immortalized keratinocytes and so open new perspectives in the therapy of skin carcinomas as well as of other skin diseases of genetic or immunological origin. © 2012 Informa

Ultrasound-Mediated Vascular Gene Transfection by Cavitation of Endothelial-Targeted Cationic Microbubbles

PubMed Central

Xie, Aris; Belcik, Todd; Qi, Yue; Morgan, Terry K.; Champaneri, Shivam A.; Taylor, Sarah; Davidson, Brian P.; Zhao, Yan; Klibanov, Alexander L.; Kuliszewski, Michael A.; Leong-Poi, Howard; Ammi, Azzdine; Lindner, Jonathan R.

2013-01-01

OBJECTIVES Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)–stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm2). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1–targeted microbubbles and by ultrasound molecular imaging of P-selectin–targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin–targeted microbubbles but was associated with

Plasmid transfection in bovine cells: Optimization using a realtime monitoring of green fluorescent protein and effect on gene reporter assay.

PubMed

Osorio, Johan S; Bionaz, Massimo

2017-08-30

Gene reporter technology (GRT) has opened several new avenues for monitoring biological events including the activation of transcription factors, which are central to the study of nutrigenomics. However, this technology relies heavily on the insertion of foreign plasmid DNA into the nuclei of cells (i.e., transfection), which can be very challenging and highly variable among cell types. The objective of this study was to investigate the optimal conditions to generate reliable GRT assay data on bovine immortalized cell lines, Madin Darby Bovine Kidney (MDBK) and bovine mammary epithelial alveolar (MACT) cells. Results are reported for two experiments. In Experiment 1, using 96 well-plate and a robotic inverted fluorescent microscope, we compared transfection efficiency among commercially available transfection reagents (TR) Lipofectamine® 3000 (Lipo3), Lipofectamine® LTX (LipoLTX), and TransIT-X2® (TransX2), three doses of TR (i.e., 0.15, 0.3, and 0.4μL/well), and three doses of Green Fluorescent Protein plasmid DNA (i.e., 10, 25, and 50ng/well). Transfection efficiency and mortality rate were analyzed using CellProfiler software. Transfection efficiency increased until the end of the experiment (20h post-transfection) at which point MACT had greater transfection than MDBK cells (16.3% vs. 2.2%). It is unclear the reason for the low transfection in MDBK cells. Maximal transfection efficiency was obtained with 0.3μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 29.5±1.9%) and 0.15μL/well of LipoLTX plus 25ng/well of plasmid DNA (ca. 4.0±0.4%) for MACT and MDBK cells, respectively. The higher amount of TR and DNA was generally associated with higher cell mortality. Using high, medium, and low transfection efficiency conditions determined in Experiment 1, we performed a GRT assay for peroxisome proliferator-activated response element (PPRE) luciferase in MACT and MDBK cells treated with 10nM or 100nM of synthetic Peroxisome Proliferator-activated Receptor

Uptake of DNA by cancer cells without a transfection reagent.

PubMed

Kong, Yanping; Zhang, Xianbo; Zhao, Yongliang; Xue, Yanfang; Zhang, Ye

2017-01-21

Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer) and THLE3 (normal liver cells) after incubation overnight by counting radioactivity of the cells' genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of "label it fluorescence in situ hybridization (FISH)" from Mirus (USA). The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA's size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA fragments directly from the environment without the aid of the transfecting

GENERATION OF TWO NOVEL CELL LINES THAT STABLY EXPRESS HAR AND FIREFLY LUCIFERASE GENES FOR ENDOCRINE SCREENING

EPA Science Inventory

Generation of Two Novel Cell Lines that Stably Express hAR and Firefly Luciferase Genes for Endocrine Screening
K.L. Bobseine*1, W.R. Kelce2, P.C. Hartig*1, and L.E. Gray, Jr.1
1USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC, 2Searle, Reproductive Toxicology Divi...

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

PubMed Central

Chu, Jun; Oh, Young-Hee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J.; Laviv, Tal; Welf, Erik S.; Dean, Kevin M.; Zhang, Feijie; Kim, Benjamin B.; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A.; Davidson, Michael W.; Kay, Mark A.; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S.; Ng, Ho-Leung; Lin, Michael Z.

2016-01-01

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin

PubMed Central

Mofford, David M.; Reddy, Gadarla Randheer; Miller, Stephen C.

2014-01-01

Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation. PMID:24616520

Use of rhenium-188 for in vivo imaging and treatment of human cervical cancer cells transfected with lentivirus expressing sodium iodide symporter.

PubMed

Zhang, Min; Shi, Shuo; Guo, Rui; Miao, Yin; Li, Biao

2016-10-01

Although survival rates for cervical cancer have improved, they need further improvement in patients with distant metastases. The sodium iodine symporter (NIS) gene has often been used in cancer therapy and imaging. We examined the therapeutic effects of rhenium-188 (188Re) in a cervical cancer xenograft model expressing the NIS gene under the control of the tumor-specific human telomerase reverse transcriptase (hTERT) promoter. We constructed two recombinant lentiviral vectors expressing enhanced green fluorescent protein (eGFP) or the NIS gene driven by the hTERT promoter. To determine the tumor-specific transcriptional activity of the hTERT promoter, the eGFP-expressing vector was stably transfected into tumor cells and normal cells. A cervical cancer HeLa cell line stably expressing NIS (HeLa-TERTNIS) was created and examined in a similar way. HeLa and HeLa-TERTNIS tumor xenografts were transplanted in nude mice, and in vivo 188Re distribution was measured using micro-SPECT/CT imaging. The therapeutic effects of 188Re were assessed over 21 days on the basis of tumor volume and the immunohistochemical findings of excised tumors. eGFP expression controlled by the hTERT promoter was substantially higher in the tumor cells than normal cells. Quantitative PCR and western blotting confirmed that HeLa-TERTNIS cells expressed high levels of NIS mRNA and protein, respectively. Further, 188Re uptake and accumulation were significantly higher in HeLa-TERTNIS cells and xenografts than HeLa cells and xenografts. In vitro and in vivo, 188Re significantly reduced the survival of HeLa-TERTNIS cells and inhibited the growth of HeLa-TERTNIS xenografts, respectively. Immunohistochemical staining showed that HeLa-TERTNIS xenograft tumors expressed higher levels of NIS and caspase-3 and lower levels of Ki-67 than HeLa xenograft tumors. Our findings indicated that hTERT promoter-driven expression of the NIS gene in HeLa cells led to 188Re uptake and therapeutic effects. Thus, NIS

Comparing visual inspection, aerobic colony counts, and adenosine triphosphate bioluminescence assay for evaluating surface cleanliness at a medical center.

PubMed

Huang, Yu-Shan; Chen, Yee-Chun; Chen, Mei-Ling; Cheng, Aristine; Hung, I-Chen; Wang, Jann-Tay; Sheng, Wang-Huei; Chang, Shan-Chwen

2015-08-01

Environmental cleaning is essential in reducing microbial colonization and health care-associated infections in hospitals. However, there is no consensus for the standard method to assess hospital cleanliness, and comparisons of newer methodology, such as adenosine triphosphate bioluminescence assay, with the traditional methods are limited. A prospective study was conducted at a medical center between January 2013 and August 2013. In each selected room, 10-12 high-touch surfaces were sampled before and after terminal cleaning. The adequacy of cleaning was evaluated by visual inspection, aerobic colony counts (ACCs), and adenosine triphosphate (ATP) bioluminescence assay. Eighty-five environmental surfaces from 8 rooms were evaluated by all 3 methods. The overall inadequacy defined by visual inspection, ACC, and ATP level was 11.8%, 20.0%, and 50.6% before cleaning and 4.7%, 5.9%, 21.2% after cleaning, respectively. A correlation between the ACC and ATP was found (r = 0.285, P < .001) using log10 values. Using ACCs <2.5 colony forming units/cm(2) as the cutoff for cleanliness, the ATP assay had better sensitivity than visual inspection (63.6% vs 27.3%). The receiver operating characteristics of the ATP assay indicated that the optimal ATP cutoff value was estimated to be 5.57 relative light units/cm(2). ATP bioluminescence assay is a sensitive and rapid tool in evaluating the quality of terminal cleaning. We emphasize the value of using a quantitative method to monitor environmental cleaning at hospitals. Copyright © 2015 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.

PubMed

Weil, D; Garçon, L; Harper, M; Duménil, D; Dautry, F; Kress, M

2002-12-01

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.

Elliptical instability in stably stratified fluid interiors

NASA Astrophysics Data System (ADS)

Vidal, J.; Hollerbach, R.; Schaeffer, N.; Cebron, D.

2016-12-01

Self-sustained magnetic fields in celestial bodies (planets, moons, stars) are due to flows in internal electrically conducting fluids. These fluid motions are often attributed to convection, as it is the case for the Earth's liquid core and the Sun. However some past or present liquid cores may be stably stratified. Alternative mechanisms may thus be needed to understand the dynamo process in these celestial objects. Turbulent flows driven by mechanical forcings, such as tides or precession, seem very promising since they are dynamo capable. However the effect of density stratification is not clear, because it can stabilize or destabilize mechanically-driven flows.To mimic an elliptical distortion due to tidal forcing in spherical geometry (full sphere and shell), we consider a theoretical base flow with elliptical streamlines and an associated density profile. It allows to keep the numerical efficiency of spectral methods in this geometry. The flow satisfies the stress-free boundary condition. We perform the stability analysis of the base state using three-dimensional simulations to study both the linear and nonlinear regimes. Stable and unstable density profiles are considered. A complementary local stability analysis (WKB) is also performed. We show that elliptical instability can still grow upon a stable stratification. We also study the mixing of the stratification by the elliptical instability. Finally we look at the dynamo capability of these flows.

The Mycobacterium marinum mel2 locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species

PubMed Central

Subbian, Selvakumar; Mehta, Parmod K; Cirillo, Suat LG; Cirillo, Jeffrey D

2007-01-01

Background Mycobacteria have developed a number of pathways that provide partial protection against both reactive oxygen species (ROS) and reactive nitrogen species (RNS). We recently identified a locus in Mycobacterium marinum, mel2, that plays a role during infection of macrophages. The molecular mechanism of mel2 action is not well understood. Results To better understand the role of the M. marinum mel2 locus, we examined these genes for conserved motifs in silico. Striking similarities were observed between the mel2 locus and loci that encode bioluminescence in other bacterial species. Since bioluminescence systems can play a role in resistance to oxidative stress, we postulated that the mel2 locus might be important for mycobacterial resistance to ROS and RNS. We found that an M. marinum mutant in the first gene in this putative operon, melF, confers increased susceptibility to both ROS and RNS. This mutant is more susceptible to ROS and RNS together than either reactive species alone. Conclusion These observations support a role for the M. marinum mel2 locus in resistance to oxidative stress and provide additional evidence that bioluminescence systems may have evolved from oxidative defense mechanisms. PMID:17239244

Use of stimulable bioluminescence from dinoflagellates as a means of detecting toxicity in the marine environment. (Reannouncement with new availability information). Professional paper

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lapota, D.; Moskowitz, G.; Grovhoug, J.

1993-03-01

Phytoplankton bioassays have been used as biological tools in assessing environmental contamination. In our laboratory, a simple bioassay has been developed which measures the light output from bioluminescence dinoflagellates for assessment of toxic effects when exposed to a single toxicant or mixture. Successful use of this type of bioassay has provided data on the acute response and has demonstrated the chronic effects, from hours up to 11 days, on dinoflagellate cells of Pyrocystis lunula and Gonyaulax polyedra upon exposure to several metals and storm drain effluent. Dinoflagellate cells were exposed to various concentrations of tributyltin chloride (TBTCI), copper (11) sulfatemore » (CUS04), zinc sulfate (ZnSO4), or storm drain effluent. Stimulable bioluminescence was measured at each test period (3 or 4 h, 24 h, 48 h, 72 h, etc.) following setup for all assays. Cells were kept in the dark for 3 or 4 h prior to testing. Stirring the cells within the chamber stimulated maximum bioluminescence from the dinoflagellates. An IC50 (an estimated concentration that is likely to cause a 50% reduction in light output) was estimated for all assays. The trend of light reduction as a response to increasing dose level of test article was observed in all assays. A reduction in light output was measured from cells exposed to 1.6, 4.2, and 12.8 ug/L TBTCI. The IC50 decreased from 8.5 ug/L at 120 h to 3.0 ug/L at 264 h. The cells exposed to 6.25%, 12.5%, and 25.0% storm drain effluent exhibited a statistically significant (P=0.05) reduction in light output in as little as 3 h exposure....Plankton, Oceanography, Bioluminescence.« less

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

PubMed Central

Herfst, Sander; Bestebroer, Theo M.; Vaes, Vincent P.; van der Hoeven, Barbara; Koster, Abraham J.; Kremers, Gert-Jan; Scott, Dana P.; Gultyaev, Alexander P.; Sorell, Erin M.; de Graaf, Miranda; Bárcena, Montserrat; Rimmelzwaan, Guus F.; Fouchier, Ron A.

2015-01-01

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses. PMID:26241861

In Vivo Bioluminescent Monitoring of Therapeutic Efficacy and Pharmacodynamic Target Assessment of Antofloxacin against Escherichia coli in a Neutropenic Murine Thigh Infection Model.

PubMed

Zhou, Yu-Feng; Tao, Meng-Ting; He, Yu-Zhang; Sun, Jian; Liu, Ya-Hong; Liao, Xiao-Ping

2018-01-01

Antimicrobial resistance among uropathogens has increased the rates of infection-related morbidity and mortality. Antofloxacin is a novel fluoroquinolone with broad-spectrum antibacterial activity against urinary Gram-negative bacilli, such as Escherichia coli This study monitored the in vivo efficacy of antofloxacin using bioluminescent imaging and determined pharmacokinetic (PK)/pharmacodynamic (PD) targets against E. coli isolates in a neutropenic murine thigh infection model. The PK properties were determined after subcutaneous administration of antofloxacin at 2.5, 10, 40, and 160 mg/kg of body weight. Following thigh infection, the mice were treated with 2-fold-increasing doses of antofloxacin from 2.5 to 80 mg/kg administered every 12 h. Efficacy was assessed by quantitative determination of the bacterial burdens in thigh homogenates and was compared with the bioluminescent density. Antofloxacin demonstrated both static and killing endpoints in relation to the initial burden against all study strains. The PK/PD index area under the concentration-time curve (AUC)/MIC correlated well with efficacy ( R 2 = 0.92), and the dose-response relationship was relatively steep, as observed with escalating doses of antofloxacin. The mean free drug AUC/MIC targets necessary to produce net bacterial stasis and 1-log 10 and 2-log 10 kill for each isolate were 38.7, 66.1, and 147.0 h, respectively. In vivo bioluminescent imaging showed a rapid decrease in the bioluminescent density at free drug AUC/MIC exposures that exceeded the stasis targets. The integration of these PD targets combined with the results of PK studies with humans will be useful in setting optimal dosing regimens for the treatment of urinary tract infections due to E. coli . Copyright © 2017 American Society for Microbiology.

[Transfection of hBcl-2 gene protects the liver against ischemia/reperfusion injury in rats during liver transplantation].

PubMed

Liu, Ji-tong; Liu, Jing-shi; Jiang, Jin-yu; Zhou, Li-xue; Liang, Gang; Li, Yan-chun

2010-12-01

To study the effect of hBcl-2 gene transfer on rat liver against ischemia-reperfusion injury, and explore the feasibility of this approach to reduce ischemia-reperfusion injury in liver transplantation. We constructed the replication-deficient recombinant adenoviruses Adv-EGFP and Adv-Bcl-2 and transfected them into 293 cells and packaged into adenovirus particles for amplification and purification. The empty plasmid vector virus was constructed similarly. Male SD rats were randomized into Adv-Bcl-2-transfected group, Adv-EGFP-transfected group, ischemia-reperfusion group, and sham-operated group, and liver allograft transplantation model was established by sleeve method. In the transfected groups, the recombinant viruses were administered by perfusion through the portal vein, and the ischemia-reperfusion and sham-operated groups received no treatment. Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expressions of bcl-2 in the liver tissue of each group, and at 0, 60 and 180 min after reperfusion, serum AST, LDH, and MDA levels were measured. Histological changes of the liver cells were evaluated by HE staining. Bcl-2 mRNA and protein expressions in Adv-Bcl-2-transfected group, as compared with those in Adv-EGFP-transfected group and control group, were significantly increased (P<0.01); the serum levels of AST, LDH and MDA in Adv-Bcl-2-transfected group were significantly lower than those of Adv-EGFP-transfected group and ischemia-reperfusion group (P<0.05 or 0.01). Compared with the sham-operated group, Adv-Bcl-2 treatment group showed lessened edema and vacuolar degeneration of the liver cells without patches or spots of necrosis. In ischemia-reperfusion and Adv-EGFP group, HE staining revealed hepatic lobular destruction and extensive liver cell swelling, enlargement, vacuolar degeneration, edema and occasional focal necrosis. Adv-Bcl-2 transfection can induce the expression of bcl-2 gene to reduce ischemia

Optimization of input parameters of acoustic-transfection for the intracellular delivery of macromolecules using FRET-based biosensors

NASA Astrophysics Data System (ADS)

Yoon, Sangpil; Wang, Yingxiao; Shung, K. K.

2016-03-01

Acoustic-transfection technique has been developed for the first time. We have developed acoustic-transfection by integrating a high frequency ultrasonic transducer and a fluorescence microscope. High frequency ultrasound with the center frequency over 150 MHz can focus acoustic sound field into a confined area with the diameter of 10 μm or less. This focusing capability was used to perturb lipid bilayer of cell membrane to induce intracellular delivery of macromolecules. Single cell level imaging was performed to investigate the behavior of a targeted single-cell after acoustic-transfection. FRET-based Ca2+ biosensor was used to monitor intracellular concentration of Ca2+ after acoustic-transfection and the fluorescence intensity of propidium iodide (PI) was used to observe influx of PI molecules. We changed peak-to-peak voltages and pulse duration to optimize the input parameters of an acoustic pulse. Input parameters that can induce strong perturbations on cell membrane were found and size dependent intracellular delivery of macromolecules was explored. To increase the amount of delivered molecules by acoustic-transfection, we applied several acoustic pulses and the intensity of PI fluorescence increased step wise. Finally, optimized input parameters of acoustic-transfection system were used to deliver pMax-E2F1 plasmid and GFP expression 24 hours after the intracellular delivery was confirmed using HeLa cells.

Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII.

PubMed

Swiech, Kamilla; Kamen, Amine; Ansorge, Sven; Durocher, Yves; Picanço-Castro, Virgínia; Russo-Carbolante, Elisa M S; Neto, Mário S A; Covas, Dimas T

2011-11-24

Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37 °C. We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34 °C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

Bioluminescence-Based Method for Measuring Assimilable Organic Carbon in Pretreatment Water for Reverse Osmosis Membrane Desalination ▿

PubMed Central

Weinrich, Lauren A.; Schneider, Orren D.; LeChevallier, Mark W.

2011-01-01

A bioluminescence-based assimilable organic carbon (AOC) test was developed for determining the biological growth potential of seawater within the reverse osmosis desalination pretreatment process. The test uses Vibrio harveyi, a marine organism that exhibits constitutive luminescence and is nutritionally robust. AOC was measured in both a pilot plant and a full-scale desalination plant pretreatment. PMID:21148685

Selective Gene Transfection of Individual Cells In Vitro with Plasmonic Nanobubbles

PubMed Central

Lukianova-Hleb, Ekaterina; Samaniego, Adam P.; Wen, Jianguo; Metelitsa, Leonid; Chang, Chung-Che; Lapotko, Dmitri

2011-01-01

Gene delivery and transfection of eukaryotic cells is widely used for research and for developing gene cell therapy. However, the existing methods lack selectivity, efficacy and safety when heterogeneous cell systems must be treated. We report a new method that employs plasmonic nanobubbles (PNBs) for delivery and transfection. A PNB is a novel, tunable cellular agent with a dual mechanical and optical action due to the formation of the vapor nanobubble around a transiently heated gold nanoparticle upon its exposure to a laser pulse. PNBs enabled the mechanical injection of the extracellular cDNA plasmid into the cytoplasm of individual target living cells, cultured leukemia cells and human CD34+CD117+ stem cells and expression of a green fluorescent protein (GFP) in those cells. PNB generation and lifetime correlated with the expression of green fluorescent protein in PNB-treated cells. Optical scattering by PNBs additionally provided the detection of the target cells and the guidance of cDNA injection at single cell level. In both cell models PNBs demonstrated a gene transfection effect in a single pulse treatment with high selectivity, efficacy and safety. Thus, PNBs provided targeted gene delivery at the single cell level in a single pulse procedure that can be used for safe and effective gene therapy. PMID:21315120

First siRNA library screening in hard-to-transfect HUVEC cells

PubMed Central

Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole UE; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra BS; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

2010-01-01

Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa® Nucleofector® 96-well Shuttle® System for siRNA screening in primary cells. Lonza's Clonetics® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME® siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector® 96-well Shuttle® System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection. PMID:20628494

Survival of bioluminescent Listeria monocytogenes and Escherichia coli O157:H7 in soft cheeses.

PubMed

Ramsaran, H; Chen, J; Brunke, B; Hill, A; Griffiths, M W

1998-07-01

Pasteurized and raw milks that had been inoculated at 10(4) cfu/ml with bioluminescent strains of Listeria monocytogenes and Escherichia coli O157:H7 were used in the manufacture of Camembert and Feta cheeses with or without nisin-producing starter culture. Survival of both organisms was determined during the manufacture and storage of Camembert and Feta cheeses at 2 +/- 1 degree C for 65 and 75 d, respectively. Bacterial bioluminescence was used as an indicator to enumerate the colonies plated on selective Listeria agar and on MacConkey agar. Escherichia coli O157:H7 survived the manufacturing process of both cheeses and was present at the end of the storage period in greater numbers than in the initial inoculum. At the end of 75 d of storage, E. coli O157:H7 was found in the brine of Feta cheese. The counts of L. monocytogenes increased as the pH of the Camembert cheese increased, and there were significant differences between the counts from samples taken from the inside and the counts from samples obtained near the surface of the cheese. The Feta cheese that contained nisin was the only cheese in which L. monocytogenes was at the level of the initial inoculum after 75 d of storage.

A serum-resistant polyamidoamine-based polypeptide dendrimer for gene transfection.

PubMed

Wu, H M; Pan, S R; Chen, M W; Wu, Y; Wang, C; Wen, Y T; Zeng, X; Wu, C B

2011-02-01

A serum tolerant polycation gene vector, G(2) PAMAM-PGlu-G(1) PAMAMs (ALA), was designed, synthesized, characterized and evaluated. A honeycomb-like molecular structure model for mechanistic explanation of ALA was postulated and discussed. Designed as a star-shaped polyamidoamine (PAMAM)-based polypeptide dendrimer through peptide bond linkages, ALA was with non-toxic low generation G(2) PAMAM (G(2)) as its central core, polyglutamate (PGlu)s as its star-shaped backbone branches and G(1) PAMAM (G(1))s as its branch grafts and peripheral terminals. IR, (1)H NMR demonstrated its successful combination. As a gene carrier, ALA exhibited good DNA binding and condensation capacity with particle size (approximately 87 nm for N/P 40, approximately 170 nm for N/P 30) and ζ-potential (approximately 16 mV for N/P 30-40), negligible cytotoxicity, exciting serum tolerant capacity and significant serum-promoted (serum-containing 56.6%>serum-free 32.7%), cell line dependent (Hek 293 > Bel 7402 > Hela), incubation period dependent (38 h > 18 h > 12 h > 9 h > 4 h > 2 h > 1 h) and sustained (peak transfection appeared at 30 h incubation) transfection efficiency. The presence of serum had not only no inhibition on, but also prominent promotion to, the transfection activity of ALA. All above features differentiated ALA clearly from most other serum-inhibitive nonviral gene carriers, and proved ALA the promising and challenging potential efficient gene vector for practical clinical application. 2010 Elsevier Ltd. All rights reserved.

Reassembly of a bioluminescent protein Renilla luciferase directed through DNA hybridization.

PubMed

Cissell, Kyle A; Rahimi, Yasmeen; Shrestha, Suresh; Deo, Sapna K

2009-01-01

Reassembly of split reporter proteins, also referred to as protein complementation, is utilized in the detection of protein-protein or protein-nucleic acid interactions. In this strategy, a reporter protein is fragmented into two inactive polypeptides to which interacting/binding partners are fused. The interaction between fused partners leads to the formation of a reassembled, active reporter. In this Communication, we have presented a proof-of-concept for the detection of a target nucleic acid sequence based on the reassembly of the bioluminescent reporter Renilla luciferase (Rluc), which is driven by DNA hybridization. Although, reassembly of Rluc though protein interactions has been demonstrated by others, the Rluc reassembly through DNA hybridization has not been shown yet, which is the novelty of this work. It is well established that bioluminescence detection offers significant advantages due to the absence of any background signal. In our study, two rationally designed fragments of Rluc were conjugated to complementary oligonucleotide probes. Hybridization of the two probes with fused Rluc fragments resulted in the reassembly of the fragments, generating active Rluc, measurable by the intensity of light given off upon addition of coelenterazine. Our study also shows that the reassembly of Rluc can be inhibited by an oligonucleotide probe that competes to bind to the hybridized probe-Rluc fragment complex, indicating a potential strategy for the quantitative detection of target nucleic acid. We were able to achieve the reassembly of Rluc fused to oligonucleotide probes using femtomole amounts of the probe-fragment protein conjugate. This concentration is approximately 4 orders of magnitude less than that reported using green fluorescent protein (GFP) as the reporter. A DNA-driven Rluc reassembly study performed in a cellular matrix did not show any interference from the matrix.

A Lithium-Air Battery Stably Working at High Temperature with High Rate Performance.

PubMed

Pan, Jian; Li, Houpu; Sun, Hao; Zhang, Ye; Wang, Lie; Liao, Meng; Sun, Xuemei; Peng, Huisheng

2018-02-01

Driven by the increasing requirements for energy supply in both modern life and the automobile industry, the lithium-air battery serves as a promising candidate due to its high energy density. However, organic solvents in electrolytes are likely to rapidly vaporize and form flammable gases under increasing temperatures. In this case, serious safety problems may occur and cause great harm to people. Therefore, a kind of lithium-air that can work stably under high temperature is desirable. Herein, through the use of an ionic liquid and aligned carbon nanotubes, and a fiber shaped design, a new type of lithium-air battery that can effectively work at high temperatures up to 140 °C is developed. Ionic liquids can offer wide electrochemical windows and low vapor pressures, as well as provide high thermal stability for lithium-air batteries. The aligned carbon nanotubes have good electric and heat conductivity. Meanwhile, the fiber format can offer both flexibility and weavability, and realize rapid heat conduction and uniform heat distribution of the battery. In addition, the high temperature has also largely improved the specific powers by increasing the ionic conductivity and catalytic activity of the cathode. Consequently, the lithium-air battery can work stably at 140 °C with a high specific current of 10 A g -1 for 380 cycles, indicating high stability and good rate performance at high temperatures. This work may provide an effective paradigm for the development of high-performance energy storage devices. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transfection of Platyhelminthes.

PubMed

Moguel, Bárbara; Bobes, Raúl J; Carrero, Julio C; Laclette, Juan P

2015-01-01

Flatworms are one of the most diverse groups within Lophotrochozoa with more than 20,000 known species, distributed worldwide in different ecosystems, from the free-living organisms in the seas and lakes to highly specialized parasites living in a variety of hosts, including humans. Several infections caused by flatworms are considered major neglected diseases affecting countries in the Americas, Asia, and Africa. For several decades, a particular interest on free-living flatworms was due to their ability to regenerate considerable portions of the body, implying the presence of germ cells that could be important for medicine. The relevance of reverse genetics for this group is clear; understanding the phenotypic characteristics of specific genes will shed light on developmental traits of free-living and parasite worms. The genetic manipulation of flatworms will allow learning more about the mechanisms for tissue regeneration, designing new and more effective anthelmintic drugs, and explaining the host-parasite molecular crosstalk so far partially inaccessible for experimentation. In this review, availability of transfection techniques is analyzed across flatworms, from the initial transient achievements to the stable manipulations now developed for free-living and parasite species.

Cyclic AMP Receptor Protein Regulates Pheromone-Mediated Bioluminescence at Multiple Levels in Vibrio fischeri ES114

PubMed Central

Lyell, Noreen L.; Colton, Deanna M.; Bose, Jeffrey L.; Tumen-Velasquez, Melissa P.; Kimbrough, John H.

2013-01-01

Bioluminescence in Vibrio fischeri ES114 is activated by autoinducer pheromones, and this regulation serves as a model for bacterial cell-cell signaling. As in other bacteria, pheromone concentration increases with cell density; however, pheromone synthesis and perception are also modulated in response to environmental stimuli. Previous studies suggested that expression of the pheromone-dependent bioluminescence activator LuxR is regulated in response to glucose by cyclic AMP (cAMP) receptor protein (CRP) (P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 164:45–50, 1985; P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 170:4040–4046, 1988; P. V. Dunlap, J. Bacteriol. 171:1199–1202, 1989; and W. F. Friedrich and E. P. Greenberg, Arch. Microbiol. 134:87–91, 1983). Consistent with this model, we found that bioluminescence in V. fischeri ES114 is modulated by glucose and stimulated by cAMP. In addition, a Δcrp mutant was ∼100-fold dimmer than ES114 and did not increase luminescence in response to added cAMP, even though cells lacking crp were still metabolically capable of producing luminescence. We further discovered that CRP regulates not only luxR but also the alternative pheromone synthase gene ainS. We found that His-tagged V. fischeri CRP could bind sequences upstream of both luxR and ainS, supporting bioinformatic predictions of direct regulation at both promoters. Luminescence increased in response to cAMP if either the ainS or luxR system was under native regulation, suggesting cAMP-CRP significantly increases luminescence through both systems. Finally, using transcriptional reporters in transgenic Escherichia coli, we elucidated two additional regulatory connections. First, LuxR-independent basal transcription of the luxI promoter was enhanced by CRP. Second, the effect of CRP on the ainS promoter depended on whether the V. fischeri regulatory gene litR was also introduced. These results suggest an integral role for CRP in pheromone signaling that

Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells.

PubMed

Hornstein, Benjamin D; Roman, Dany; Arévalo-Soliz, Lirio M; Engevik, Melinda A; Zechiedrich, Lynn

2016-01-01

The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery.

Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells